Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome

Dying cells are capable of activating the innate immune system and inducing a sterile inflammatory response. Here, we show that necrotic cells are sensed by the Nlrp3 inflammasome resulting in the subsequent release of the proinflammatory cytokine IL-1β. Necrotic cells produced by pressure disruption, hypoxic injury, or complement-mediated damage were capable of activating the Nlrp3 inflammasome. Nlrp3 inflammasome activation was triggered in part through ATP produced by mitochondria released from damaged cells. Neutrophilic influx into the peritoneum in response to necrotic cells in vivo was also markedly diminished in the absence of Nlrp3. Nlrp3-deficiency moreover protected animals against mortality, renal dysfunction, and neutrophil influx in an in vivo renal ischemic acute tubular necrosis model. These findings suggest that the inhibition of Nlrp3 inflammasome activity can diminish the acute inflammation and damage associated with tissue injury.     

Cloning of α-β fusion gene from Clostridium perfringens and its expression

AIM: To study the cloning of alpha-beta fusion gene from Clostridium perfringens and the immunogenicity of alpha-beta fusion expression. METHODS: Cloning was accomplished after PCR amplification from strains NCTC64609 and C58-1 of the protective antigen genes of alpha-toxin and beta-toxin. The fragment of the gene was cloned using plasmid pZCPAB. This fragment coded for the gene with the stable expression of alpha-beta fusion gene binding. In order to verify the exact location of the alpha-beta fusion gene, domain plasmids were constructed. The two genes were fused into expression vector pBV221. The expressed alpha-beta fusion protein was identified by ELISA, SDS-PAGE, Western blotting and neutralization assay. RESULTS: The protective alpha-toxin gene (cpa906) and the beta-toxin gene (cpb930) were obtained. The recombinant plasmid pZCPAB carrying alpha-beta fusion gene was constructed and transformed into BL21(DE3). The recombinant strain BL21(DE3)(pZCPAB) was obtained. After the recombinant strain BL21(DE3)(pZCPAB) was induced by 42 degC, its expressed product was about 22.14% of total cellular protein at SDS-PAGE and thin-layer gel scanning analysis. Neutralization assay indicated that the antibody induced by immunization with alpha-beta fusion protein could neutralize the toxicity of alpha-toxin and beta-toxin. CONCLUSION: The obtained alpha-toxin and beta-toxin genes are correct. The recombinant strain BL21(DE3)(pZCPAB) could produce alpha-beta fusion protein. This protein can be used for immunization and is immunogenic. The antibody induced by immunization with alpha-beta fusion protein could neutralize the toxicity of alpha-toxin and beta-toxin.     

microRNA 17/20 inhibits cellular invasion and tumor metastasis in breast cancer by heterotypic signaling

microRNAs are thought to regulate tumor progression and invasion via direct interaction with target genes within cells. Here the microRNA17/20 cluster is shown to govern cellular migration and invasion of nearby cells via heterotypic secreted signals. microRNA17/20 abundance is reduced in highly invasive breast cancer cell lines and node-positive breast cancer specimens. Cell-conditioned medium from microRNA17/20–overexpressing noninvasive breast cancer cell MCF7 was sufficient to inhibit MDA-MB-231 cell migration and invasion through inhibiting secretion of a subset of cytokines, and suppressing plasminogen activation via inhibition of the secreted plasminogen activators (cytokeratin 8 and α-enolase). microRNA17/20 directly repressed IL-8 by targeting its 3′ UTR, and inhibited cytokeratin 8 via the cell cycle control protein cyclin D1. At variance with prior studies, these results demonstrated a unique mechanism of how the altered microRNA17/20 expression regulates cellular secretion and tumor microenvironment to control migration and invasion of neighboring cells in breast cancer. These findings not only reveal an antiinvasive function of miR-17/20 in breast cancer, but also identify a heterotypic secreted signal that mediates the microRNA regulation of tumor metastasis.     

N-terminal domain of complexin independently activates calcium-triggered fusion

Complexin activates Ca²⁺-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca²⁺-sensor synaptotagmin. The N-terminal domain of complexin is important for activation, but its molecular mechanism is still poorly understood. Here, we observed that a split pair of N-terminal and central domain fragments of complexin is sufficient to activate Ca²⁺-triggered release using a reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery. The N-terminal domain can also interact independently with membranes, which is enhanced by a cooperative interaction with the neuronal SNARE complex. We show by mutagenesis that membrane binding of the N-terminal domain is essential for activation of Ca²⁺-triggered fusion. Consistent with the membrane-binding property, the N-terminal domain can be substituted by the influenza virus hemagglutinin fusion peptide, and this chimera also activates Ca²⁺-triggered fusion. Membrane binding of the N-terminal domain of complexin therefore cooperates with the other fusogenic elements of the synaptic fusion machinery during Ca²⁺-triggered release.Neurotransmitter release occurs upon fusion of synaptic vesicles with the plasma membrane (1, 2). Synaptic vesicle fusion is orchestrated by the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) fusion proteins (3, 4), in conjunction with synaptotagmin, complexin, and other synaptic proteins. The Ca²⁺-sensor synaptotagmin is essential for Ca²⁺-triggered release (5–8). Neuronal SNARE proteins form a ternary complex consisting of synaptobrevin/vesicle-associated membrane protein (VAMP2), syntaxin, and synaptosomal-associated protein 25 (SNAP-25). The main isoform synaptotagmin-1 is involved in synchronous release, and forms a conserved Ca²⁺-independent interface with the ternary SNARE complex (9), along with Ca²⁺-dependent interactions with the plasma membrane, and potentially other interfaces with the SNARE complex (10). Complexin is a small cytosolic α-helical protein abundant in the presynaptic terminal (11) that interacts with the SNARE complex (12) and the membrane (13).Complexin has at least two functions: It “activates” (i.e., greatly enhances) Ca²⁺-triggered synchronous neurotransmitter release by cooperating with synaptotagmin, and it regulates spontaneous release in the presynaptic terminal (recently reviewed in refs. 14–16). The activating function of complexin is conserved across all species (mammals, Drosophila, and Caenorhabditis elegans) and different types of Ca²⁺-triggered synaptic vesicle fusion studied to date (11, 17–26). Complexin also regulates spontaneous neurotransmitter release, although this effect is less conserved among species and varies depending on experimental conditions: for example, in Drosophila, spontaneous release increases with knockout of complexin (27, 28). Likewise, knockdown of complexin in cultured cortical neurons increases spontaneous release, although knockout of complexin in mice only affects spontaneous release depending on the particular neuronal cell type (20, 23, 24, 29). Exactly how complexin can exhibit these dual effects on Ca²⁺-triggered and spontaneous synaptic vesicle fusion remains enigmatic; however, it is known that different domains of complexin play different roles in Ca²⁺-triggered and spontaneous vesicle fusion, as summarized in the following paragraphs.Here, we focus on the complexin-1 isoform (referred to as Cpx in the following). Cpx can be divided into four domains (Fig. 1A, Bottom) that are involved in different functions. The N-terminal domain (residues 1–27) of Cpx is important for activation of synchronous Ca²⁺-triggered release in murine neurons (20, 30, 31) and in isolated chromaffin cells (32). However, N-terminal truncation of Cpx in C. elegans neuromuscular junctions does not decrease Ca²⁺-triggered release, but rather increases spontaneous release (21, 22), perhaps suggesting that reduction of activation may have been masked by a simultaneous increase of spontaneous fusion in these previous experiments.Open in a separate windowFig. 1.Cpx (26–83) fragment reduces spontaneous fusion similar to wild-type Cpx. (A) Schematic diagram of the single-vesicle content mixing assay (35) (Methods) and domain diagrams of Cpx and Cpx fragments used in this figure. PM, vesicles with reconstituted syntaxin-1A and SNAP-25A that mimic the plasma membrane; SV, vesicles with reconstituted synaptobrevin-2 and synaptotagmin-1 that mimic synaptic vesicles. The bar graphs show the effects of 2 μM Cpx or Cpx fragments on the SV/PM vesicle association count during the first acquisition periods (Methods) (B), the average probability of spontaneous fusion events per second (C), the amplitude of the first 1-s time bin (probability of a fusion event in that bin) upon Ca²⁺ injection (D), and the decay rate (1/τ) of the histogram upon Ca²⁺ injection (E). The fusion probabilities and amplitudes were normalized with respect to the corresponding number of analyzed SV/PM vesicle pairs (Methods). Individual histograms are in Figs. S1 and ​andS2.S2. The error bars in B–D are SDs for multiple independent repeat experiments (20, 29, 30, 33–38). Although the accessory domain is required for regulating spontaneous release, mutations of this domain do not affect the activating function of Cpx for Ca²⁺-triggered release compared with wild-type neurons in rescue experiments of Cpx knockdown (23, 39).The central domain of Cpx (residues 49–70) is essential for all functions of complexins in all species studied to date, including priming (23, 24, 39, 40), inhibiting spontaneous release (18, 20–22, 35, 37, 38), and activation of Ca²⁺-triggered release (17, 18, 20, 22, 30, 31, 35, 41).The C-terminal domain binds to phospholipids (24, 42), and it is important for vesicle priming in neurons (24, 32, 43). Moreover, Cpx without the C-terminal domain does not reduce spontaneous release in neuronal cultures, but it still activates Ca²⁺-triggered release in neuronal cultures (24) and in a reconstituted system (35). The C-terminal domain is sensitive to membrane curvature, and it may thus localize Cpx to the synaptic membrane (13, 44).Structurally, in isolation, both the N- and C-terminal domains of Cpx are largely flexible, although the accessory and central domains have α-helical propensity (45). The α-helical central domain of Cpx binds to the groove between the synaptobrevin-2 and syntaxin-1A α-helices in the center of the neuronal SNARE complex (12, 46). Cpx has two conformations when bound to the ternary SNARE complex, one of which induces a conformational change at the membrane-proximal C-terminal end of the ternary SNARE complex that specifically depends on the N-terminal, accessory, and central domains of Cpx (47).Cpx has been studied extensively with reconstituted systems (35, 38, 48–52). The single-vesicle fusion assay described by Lai et al. (35) qualitatively reproduced the effects of synaptotagmin-1 and Cpx in both spontaneous and Ca²⁺-triggered release that have been observed in cortical neuronal cultures (9, 35).Here, we conducted single-vesicle fusion and single-molecule membrane-binding experiments to obtain new insights into the function of the Cpx N-terminal domain. We found that the N-terminal domain can be physically separated from the accessory and central domains of Cpx and still preserve its role in activating Ca²⁺-triggered release. The N-terminal domain interacts with membranes, an interaction that is enhanced by the presence of SNARE complex. Moreover, the N-terminal domain of full-length Cpx can be functionally substituted by the fusion peptide of influenza virus hemagglutinin (HA), suggesting that similar fusion elements and principles are used in different contexts of biological membrane fusion.     

Transplantation of human islets without immunosuppression

Transplantation of pancreatic islets is emerging as a successful treatment for type-1 diabetes. Its current stringent restriction to patients with critical metabolic lability is justified by the long-term need for immunosuppression and a persistent shortage of donor organs. We developed an oxygenated chamber system composed of immune-isolating alginate and polymembrane covers that allows for survival and function of islets without immunosuppression. A patient with type-1 diabetes received a transplanted chamber and was followed for 10 mo. Persistent graft function in this chamber system was demonstrated, with regulated insulin secretion and preservation of islet morphology and function without any immunosuppressive therapy. This approach may allow for future widespread application of cell-based therapies.The transplantation of isolated islets of Langerhans has evolved into a successful method to restore endogenous insulin secretion and stabilize glycemic control without the risk of hypoglycemia (1, 2). However, due to persistent lack of human donor pancreata and the requirement of chronic immune suppression to prevent graft rejection through allo- and autoimmunity, the indication for islet transplantation is restricted to patients with complete insulin deficiency, critical metabolic lability, and repeated severe hypoglycemia despite optimal diabetes management and compliance (3). Furthermore, progressive loss of islet function over time due to chronic hypoxia and inflammatory processes at the intraportal transplantation site remain additional unresolved challenges in islet transplantation (4, 5).When islets are immune-isolated, the lack of oxygen impairs the survival and long-term function of the cells. Experimental approaches to overcome this impediment have involved the implantation of hypoxia-resistant islets, stimulation and sprouting of vessels, and the use of islets designed to contain an intracellular oxygen carrier as well as local oxygen production by electrochemical processes or photosynthesis (6). However, so far, none of these methods have been capable of guaranteeing an adequate physiological oxygen concentration or to allow, at the same time, an adequate immunoprotective environment. To overcome these major obstacles, we have developed a strategy for islet macroencapsulation that provides sufficient immune isolation and permits endogenously regulated islet graft function. Here we demonstrate a system that allows a controlled oxygen supply to the islet graft by means of an integrated oxygen reservoir that can be refilled regularly and can maintain oxygen pressure. Earlier we demonstrated that a sufficient supply of oxygen for maintaining optimal islet function can simultaneously ensure functional potency and immunoprotective characteristics of the device. After application of this bioartificial pancreas system in allogeneic and xenogeneic preclinical diabetes models (7–9) the method was then applied to allogeneic human islet transplantation in an individual treatment approach in a patient with long-term type-1 diabetes. The objective of this study was to determine whether the islet allograft could survive over a prolonged follow-up period, without any immunosuppressive therapy, and could maintain glucose responsiveness. Furthermore, biocompatibility of the macrocapsule and the practicability of the oxygen refilling procedure in daily life were evaluated.     

GABARAPs regulate PI4P-dependent autophagosome:lysosome fusion

The Atg8 autophagy proteins are essential for autophagosome biogenesis and maturation. The γ-aminobutyric acid receptor-associated protein (GABARAP) Atg8 family is much less understood than the LC3 Atg8 family, and the relationship between the GABARAPs’ previously identified roles as modulators of transmembrane protein trafficking and autophagy is not known. Here we report that GABARAPs recruit palmitoylated PI4KIIα, a lipid kinase that generates phosphatidylinositol 4-phosphate (PI4P) and binds GABARAPs, from the perinuclear Golgi region to autophagosomes to generate PI4P in situ. Depletion of either GABARAP or PI4KIIα, or overexpression of a dominant-negative kinase-dead PI4KIIα mutant, decreases autophagy flux by blocking autophagsome:lysosome fusion, resulting in the accumulation of abnormally large autophagosomes. The autophagosome defects are rescued by overexpressing PI4KIIα or by restoring intracellular PI4P through “PI4P shuttling.” Importantly, PI4KIIα’s role in autophagy is distinct from that of PI4KIIIβ and is independent of subsequent phosphatidylinositol 4,5 biphosphate (PIP₂) generation. Thus, GABARAPs recruit PI4KIIα to autophagosomes, and PI4P generation on autophagosomes is critically important for fusion with lysosomes. Our results establish that PI4KIIα and PI4P are essential effectors of the GABARAP interactome’s fusion machinery.Macroautophagy (autophagy) is orchestrated by multiple autophagy-related (Atg) proteins (1). Among these, the Atg8 proteins are essential for autophagosome biogenesis and maturation. Mammals have at least six Atg8 orthologs that can be broadly classified into two large subfamilies: LC3s (light-chain 3) and GABARAPs (γ-aminobutyric acid receptor-associated proteins)/GATE-16s (Golgi-associated ATPase enhancer of 16 kDa), hereafter referred to collectively as GABARAPs. GABARAPs were initially identified as trafficking modulators for transmembrane receptors from the Golgi to the plasma membrane (2), and subsequently as Atg8s (1). Functional studies in mammalian cells have placed GABARAPs downstream of LC3 during autophagy (3).The complexity of the mammalian Atg8 protein network was highlighted by a recent screen that revealed a cohort of at least 67 binding partners, a third of which are unique to either the LC3 or GABARAP families (4). Phosphatidylinositol 4-kinase IIα (PI4KIIα), which generates phosphatidylinositol 4-phosphate (PI4P) from phosphatidylinositol, was identified as a binding partner for GABARAPs, but not for LC3 (4). PI4KIIα is one of four vertebrate PI4Ks (5), and it has not been previously implicated in autophagy. Here we establish for the first time, to our knowledge, that GABARAPs govern the fusion of autophagosomes with lysosomes (A:L fusion) through PI4KIIα-mediated in situ PI4P generation on autophagosomes. We propose a working model that integrates GABARAPs’ critical roles as trafficking modulators and autophagy effectors through PI4KIIα.     

Properties of Slo1 K+ channels with and without the gating ring

High-conductance Ca²⁺- and voltage-activated K⁺ (Slo1 or BK) channels (KCNMA1) play key roles in many physiological processes. The structure of the Slo1 channel has two functional domains, a core consisting of four voltage sensors controlling an ion-conducting pore, and a larger tail that forms an intracellular gating ring thought to confer Ca²⁺ and Mg²⁺ sensitivity as well as sensitivity to a host of other intracellular factors. Although the modular structure of the Slo1 channel is known, the functional properties of the core and the allosteric interactions between core and tail are poorly understood because it has not been possible to study the core in the absence of the gating ring. To address these questions, we developed constructs that allow functional cores of Slo1 channels to be expressed by replacing the 827-amino acid gating ring with short tails of either 74 or 11 amino acids. Recorded currents from these constructs reveals that the gating ring is not required for either expression or gating of the core. Voltage activation is retained after the gating ring is replaced, but all Ca²⁺- and Mg²⁺-dependent gating is lost. Replacing the gating ring also right-shifts the conductance-voltage relation, decreases mean open-channel and burst duration by about sixfold, and reduces apparent mean single-channel conductance by about 30%. These results show that the gating ring is not required for voltage activation but is required for Ca²⁺ and Mg²⁺ activation. They also suggest possible actions of the unliganded (passive) gating ring or added short tails on the core.Slo1 channels are expressed in most human tissues and play key roles in many important physiological processes, including smooth muscle contraction, neurotransmitter release, neuronal excitability, hair cell tuning, and action potential termination (1–6). Slo1 channels also are named BK (Big K⁺) or MaxiK channels because of their high single-channel conductance (∼300 pS in 150-mM symmetrical K⁺). Slo1 channels are activated synergistically by both depolarization and intracellular calcium (7–9), linking these two activators in a negative feed-back system to restore negative membrane potential which, in turn, closes voltage-activated Ca²⁺ channels. The dual regulation by voltage and calcium led Hille (10) to predict that BK channels function like the classical Hodgkin–Huxley delayed rectifier channel, except that the range of voltage activation was set by the intracellular Ca²⁺ concentration. The cloning (11) and analysis of the Slo1 channel structure seemed to validate this prediction, in that Slo1 appeared to be modular in its construction, having a core domain containing a voltage sensor controlling a K⁺-selective pore and a long C-terminal tail forming a gating ring structure comprised of four pairs of regulators of the conductance of K⁺ (RCK) domains for sensing and transducing the effect of Ca²⁺ binding to the core.One of the four identical α subunits that assemble to form the Slo1 WT channel (Slo1-WT) is shown in Fig. 1 Top. For the mbr5 cDNA (12) used in this study, the “core” consists of 342 residues including seven transmembrane segments (S0–S6) and the S6–RCK1 linker sequence, which is attached to a long tail of 827 residues. The tail sequence of Slo1-WT is distinct from the cytoplasmic domains of other members of the K⁺ channel extended family. Structure–function studies of the tail have shown the existence of two high-affinity Ca²⁺ binding sites (13, 14) and one low-affinity Mg²⁺ site (14, 15). Modulation of the channel also occurs by additional biological factors, including protons, heme, carbon monoxide, phosphorylation, and oxidation (16–20), all of which may function via their interaction with the tail. Thus, the large tail accommodates a variety of regulatory domains which sense different intracellular factors, leading to pushing or tugging against the core to facilitate or inhibit channel gating. These complicated allosteric interactions between core and tail almost certainly involve several transduction pathways (21–23), all of which alter the properties of the core. Thus, a logical starting point to begin investigating the allosteric interactions would be to understand the baseline properties of the isolated core. However, this approach has been hampered by the inability to express functional cores in the absence of the tail. Previous analysis of truncated expression constructs of Slo1 channels found that their processing stalls in the endoplasmic reticulum (ER), they are not assembled into tetramers, they fail to be exported to the plasma membrane, or they are nonfunctional (24). We now show that core constructs without gating rings can be expressed by leaving a short region required for subunit tetramerization and by appending a small tail domain which facilitates processing and efficient export to the plasma membrane. Thus, we now are able to investigate gating in the absence of a gating ring.Open in a separate windowFig. 1.Slo1 channel constructs used in this study. The Slo1 channel constructs used in this study are based on the mouse mbr5 cDNA (12) and the mouse Shaker family Kv1.4 channel (25). The “Slo1 core and tail” refers to the first 342 and the last 827 amino acid residues. The “Kv1.4 tail” refers to the last 74 amino acid residues of Kv1.4. The different channel constructs are designated as follows: Slo1-WT is Slo1 full-length WT; Slo1C-KvT is a Slo1 core with a 74-residue Kv1.4 tail; Slo1C-Kv-minT is a Slo1 core with a Kv1.4 11-residue mini tail; Slo1C-KvT_NAFQ is a Slo1 core with a 74-residue Kv1.4 tail with NAFQ substituted for KKFR in the tail; Slo1C-KvT R207E is Slo1C-KvT with R207E in S4 in the core.     

The Studies on α-Pinene Oxidation over the TS-1. The Influence of the Temperature,Reaction Time,Titanium and Catalyst Content

The work presents the results of studies on α-pinene oxidation over the TS-1 catalysts with different Ti content (in wt%): TS-1_1 (9.92), TS-1_2 (5.42), TS-1_3 (3.39) and TS-1_4 (3.08). No solvent was used in the oxidation studies, and molecular oxygen was used as the oxidizing agent. The effect of titanium content in the TS-1 catalyst, temperature, reaction time and amount of the catalyst in the reaction mixture on the conversion of α-pinene and the selectivities of appropriate products was investigated. It was found that it is most advantageous to carry out the process of α-pinene oxidation in the presence of the TS-1 catalyst with the titanium content of 5.42 wt% (TS-1_2), at the temperature of 85 °C, for 6 h and with the catalyst TS-1 content in the reaction mixture of 1 wt%. Under these conditions the conversion of α-pinene amounted to 34 mol%, and the selectivities of main products of α-pinene oxidation process were: α-pinene oxide (29 mol%), verbenol (15 mol%) and verbenone (12 mol%). In smaller quantities also campholenic aldehyde, trans-pinocarveol, myrtenal, myrtenol, L-carveol, carvone and 1,2-pinanediol were also formed. These products are of great practical importance in food, cosmetics, perfumery and medicine industries. Kinetic studies were also performed for the studied process.     

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor

Unlike conventional αβ T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1⁺ and Vδ2⁺ γδ TCR-mediated ligand recognition, the mode of Vδ3⁺ TCR ligand engagement is unknown. MHC class I–related protein, MR1, presents vitamin B metabolites to αβ T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2⁻ γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3⁺ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3⁺ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Characterized by both innate and adaptive immune cell functions, γδ T cells are an unconventional T cell subset. While the functional role of γδ T cells is yet to be fully established, they can play a central role in antimicrobial immunity (1), antitumor immunity (2), tissue homeostasis, and mucosal immunity (3). Owing to a lack of clarity on activating ligands and phenotypic markers, γδ T cells are often delineated into subsets based on the expression of T cell receptor (TCR) variable (V) δ gene usage, grouped as Vδ2⁺ or Vδ2⁻.The most abundant peripheral blood γδ T cell subset is an innate-like Vδ2⁺subset that comprises ∼1 to 10% of circulating T cells (4). These cells generally express a Vγ9 chain with a focused repertoire in fetal peripheral blood (5) that diversifies through neonatal and adult life following microbial challenge (6, 7). Indeed, these Vγ9/Vδ2⁺ T cells play a central role in antimicrobial immune response to Mycobacterium tuberculosis (8) and Plasmodium falciparum (9). Vγ9/Vδ2⁺ T cells are reactive to prenyl pyrophosphates that include isopentenyl pyrophosphate and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (8) in a butyrophilin 3A1- and BTN2A1-dependent manner (10–13). Alongside the innate-like protection of Vγ9/Vδ2⁺ cells, a Vγ9⁻ population provides adaptive-like immunobiology with clonal expansions that exhibit effector function (14).The Vδ2⁻ population encompasses the remaining γδ T cells but most notably the Vδ1⁺ and Vδ3⁺ populations. Vδ1⁺ γδ T cells are an abundant neonatal lineage that persists as the predominating subset in adult peripheral tissue including the gut and skin (15–18). Vδ1⁺ γδ T cells display potent cytokine production and respond to virally infected and cancerous cells (19). Vδ1⁺ T cells were recently shown to compose a private repertoire that diversifies, from being unfocused to a selected clonal TCR pool upon antigen exposure (20–23). Here, the identification of both Vδ1⁺ T_naive and Vδ1⁺ T_effector subsets and the Vδ1⁺ T_naive to T_effector differentiation following in vivo infection point toward an adaptive phenotype (22).The role of Vδ3⁺ γδ T cells has remained unclear, with a poor understanding of their lineage and functional role. Early insights into Vδ3⁺ γδ T cell immunobiology found infiltration of Vδ3⁺ intraepithelial lymphocytes (IEL) within the gut mucosa of celiac patients (24). More recently it was shown that although Vδ3⁺ γδ T cells represent a prominent γδ T cell component of the gut epithelia and lamina propria in control donors, notwithstanding pediatric epithelium, the expanding population of T cells in celiac disease were Vδ1⁺ (25). Although Vδ3⁺ IELs compose a notable population of gut epithelia and lamina propria T cells (∼3 to 7%), they also formed a discrete population (∼0.2%) of CD4⁻CD8⁻ T cells in peripheral blood (26). These Vδ3⁺ DN γδ T cells are postulated to be innate-like due to the expression of NKG2D, CD56, and CD161 (26). When expanded in vitro, these cells degranulated and killed cells expressing CD1d and displayed a T helper (Th) 1, Th2, and Th17 response in addition to promoting dendritic cell maturation (26). Peripheral Vδ3⁺ γδ T cells frequencies are known to increase in systemic lupus erythematosus patients (27, 28), and upon cytomegalovirus (29) and HIV infection (30), although, our knowledge of their exact role and ligands they recognize remains incomplete.The governing paradigms of antigen reactivity, activation principles, and functional roles of γδ T cells remain unresolved. This is owing partly due to a lack of knowledge of bona fide γδ T cell ligands. Presently, Vδ1⁺ γδ T cells remain the best characterized subset with antigens including Major Histocompatibility Complex (MHC)-I (31), monomorphic MHC-I–like molecules such as CD1b (32), CD1c (33), CD1d (34), and MR1 (35), as well as more diverse antigens such as endothelial protein coupled receptor (EPCR) and phycoerythrin (PE) (36, 37). The molecular determinants of this reactivity were first established for Vδ1⁺ TCRs in complex with CD1d presenting sulfatide (38) and α-galactosylceramide (α-GalCer) (34), which showed an antigen-dependent central focus on the presented lipids and docked over the antigen-binding cleft.In humans, mucosal-associated invariant T (MAIT) cells are an abundant innate-like αβ T cell subset typically characterized by a restricted TCR repertoire (39–43) and reactivity to the monomorphic molecule MR1 presenting vitamin B precursors and drug-like molecules of bacterial origin (41, 44–46). Recently, populations of atypical MR1-restricted T cells have been identified in mice and humans that utilize a more diverse TCR repertoire for MR1-recognition (42, 47, 48). Furthermore, MR1-restricted γδ T cells were identified in blood and tissues including Vδ1⁺, Vδ3⁺, and Vδ5⁺ clones (35). As seen with TRAV 1-2⁻, unconventional MAITs cells the isolated γδ T cells exhibited MR1-autoreactivity with some capacity for antigen discrimination within the responding compartment (35, 48). Structural insight into one such MR1-reactive Vδ1⁺ γδ TCR showed a down-under TCR engagement of MR1 in a manner that is thought to represent a subpopulation of MR1-reactive Vδ1⁺ T cells (35). However, biochemical evidence suggested other MR1-reactive γδ T cell clones would likely employ further unusual docking topologies for MR1 recognition (35).Here, we expanded our understanding of a discrete population of human Vδ3⁺ γδ T cells that display reactivity to MR1. We provide a molecular basis for this Vδ3⁺ γδ T cell reactivity and reveal a side-on docking for MR1 that is distinct from the previously determined Vδ1⁺ γδ TCR-MR1-Ag complex. A Vδ3⁺ γδ TCR does not form contacts with the bound MR1 antigen, and we highlight the importance of non–germ-line Vδ3 residues in driving this MR1 restriction. Accordingly, we have provided key insights into the ability of human γδ TCRs to recognize MR1 in an antigen-independent manner by contrasting mechanisms.     

Thymus leukemia antigen controls intraepithelial lymphocyte function and inflammatory bowel disease

Intestinal intraepithelial lymphocytes (IEL) bear a partially activated phenotype that permits them to rapidly respond to antigenic insults. However, this phenotype also implies that IEL must be highly controlled to prevent misdirected immune reactions. It has been suggested that IEL are regulated through the interaction of the CD8αα homodimer with the thymus leukemia (TL) antigen expressed by intestinal epithelial cells. We have generated and characterized mice genetically-deficient in TL expression. Our findings show that TL expression has a critical role in maintaining IEL effector functions. Also, TL deficiency accelerated colitis in a genetic model of inflammatory bowel disease. These findings reveal an important regulatory role of TL in controlling IEL function and intestinal inflammation.     

Biotic homogenization can decrease landscape-scale forest multifunctionality

Many experiments have shown that local biodiversity loss impairs the ability of ecosystems to maintain multiple ecosystem functions at high levels (multifunctionality). In contrast, the role of biodiversity in driving ecosystem multifunctionality at landscape scales remains unresolved. We used a comprehensive pan-European dataset, including 16 ecosystem functions measured in 209 forest plots across six European countries, and performed simulations to investigate how local plot-scale richness of tree species (α-diversity) and their turnover between plots (β-diversity) are related to landscape-scale multifunctionality. After accounting for variation in environmental conditions, we found that relationships between α-diversity and landscape-scale multifunctionality varied from positive to negative depending on the multifunctionality metric used. In contrast, when significant, relationships between β-diversity and landscape-scale multifunctionality were always positive, because a high spatial turnover in species composition was closely related to a high spatial turnover in functions that were supported at high levels. Our findings have major implications for forest management and indicate that biotic homogenization can have previously unrecognized and negative consequences for large-scale ecosystem multifunctionality.It is widely established that high local-scale biodiversity increases levels of individual ecosystem functions in experimental ecosystems (1–4), and that biodiversity is even more important for the simultaneous maintenance of multiple functions at high levels (i.e., ecosystem multifunctionality) (5–8). Because the capacity of natural ecosystems to maintain multiple functions and services is crucial for human well-being (9), the positive diversity–multifunctionality relationship is often used as an argument to promote biodiversity conservation (6, 10). However, although society seeks to maximize the delivery of potentially conflicting ecosystem services, such as food production, bioenergy generation, and carbon storage at the landscape scale (11–13), research into the relationship between biodiversity and ecosystem multifunctionality has been largely limited to local-scale studies, where diversity is manipulated in experimental plant communities. Although some studies have focused on more natural communities distributed over larger spatial extents (e.g., 14–16), they examined relationships between local-scale biodiversity and local-scale multifunctionality. The only previous study to investigate multifunctionality at larger scales (17) simulated artificial landscapes using data from experimental grassland communities. It showed that although different aspects of biodiversity affected multifunctionality, local-scale (α-) diversity was a much stronger driver than the turnover of species between sites (β-diversity). However, whether those findings can be extrapolated to real-world (i.e., natural, seminatural) ecosystems, such as forests, is unknown. As a result, we have a poor understanding of how multifunctionality relates to biodiversity at the larger spatial scales that are most relevant to ecosystem managers. This question is of particular concern, given recent findings suggesting that human-driven homogenization of communities [loss of β-diversity (18–21)] may be just as widespread as α-diversity declines (22, 23).Multifunctionality can be measured by a variety of methods, and the most appropriate means of doing so remains unresolved (24–27), particularly at larger scales, where the desired distribution of ecosystem function across the landscape has not been quantified. At local scales, one can quantify ecosystem multifunctionality as the number of ecosystem functions that exceed a given threshold value, where the threshold equals a certain percentage of the maximum observed value of each function (10, 24) (hereafter “threshold-based multifunctionality”; Fig. 1B). This threshold reflects the minimum value of ecosystem functioning that is deemed satisfactory. Because trade-offs between ecosystem functions or services are commonplace (5, 7, 28, 29), it is often impossible to maximize all of the desired functions in a local community (6). However, when different species provide different functions (5, 7) at larger spatial scales, a high spatial turnover in community composition (i.e., a high β-diversity) across the landscape can cause different parts of the landscape to provide different functions at high levels (defined as high threshold-based β-multifunctionality; Fig. 1B). Therefore, high β-diversity might cause all desired ecosystem functions to be provided at high levels in at least one patch within a landscape [and hence promote threshold-based landscape-scale or γ-multifunctionality (30)] (Fig. 1B), but only if (i) species differ in the functions they support and (ii) there is no “superspecies” that supports the majority of functions. This threshold-based γ-multifunctionality may be relevant for cases where forest landscapes are managed for many different services (e.g., timber production, limitation of nutrient runoff, ecotourism), but where each of these services only needs to be provided at high levels in a part of the landscape, not everywhere (31). Alternatively, a manager may seek to promote the total delivery of many summed individual ecosystem functions across a landscape. We define this total delivery as sum-based γ-multifunctionality (Fig. 1B). This metric may be a more appropriate measure of multifunctionality in cases where the benefits of ecosystem services are manifested at large scales, such as carbon sequestration or water purification (32). In this case, β-diversity might only promote sum-based γ-multifunctionality if nonadditive diversity effects, such as resource partitioning, species-environment matching, or spillover effects, operate at relatively large spatial scales (33, 34). It is therefore likely that the importance of β-diversity for γ-multifunctionality varies depending on the desired pattern of ecosystem service provision.Open in a separate windowFig. 1.Quantifying biodiversity and multifunctionality across spatial scales. The light yellow areas represent hypothetical landscapes, consisting of (white) local communities. In these communities, some species are present (colored icons in A and C), whereas others are absent (gray icons). Similarly, some functions are performing above a hypothetical threshold of 0.5 (colored icons in B), whereas others are not (gray icons). Diversity and threshold-based multifunctionality are quantified at (i) the local plot (α-) scale as the number of species present (two and three in A) or functions performing above a given threshold (two and three in B), (ii) the β-scale as the turnover in species composition [ = 1 ? log((a + b + 2c)/(a + b + c)) = 1 ? log((1 + 2 + 2)/(1 + 2 + 1)) = 0.90 in A (49)] or functions [ = 1 ? log((a + b + 2c)/(a + b + c)) = 1 ? log((1 + 2 + 2)/(1 + 2 + 1)) = 0.90 in B (49)] across plots, and (iii) the landscape (γ-) scale as the number of functions (four in B) present in at least one plot. Sum-based γ-multifunctionality is defined as the sum of all standardized ecosystem values in a landscape (= 0.8 + 0.2 + 0.7 + 0.4 + 0.9 + 1.0 + 0.1 + 0.6 = 4.7). In contrast to threshold-based multifunctionality, sum-based multifunctionality is not analogous to biodiversity (where species are either present or absent), and can therefore not be partitioned into α- or β-components. (C) This framework allows investigation of whether γ-multifunctionality is promoted by α- and/or β-diversity.Forests provide many ecosystem services, including wood production, regulation of water quality and climate, and recreation (35, 36). Most present-day European forests and almost all forest plantations worldwide are dominated by only one or a few tree species (15, 37), although their diversity could be promoted relatively easily by planting more species or by encouraging natural regeneration. This fact makes the understanding of diversity–multifunctionality relationships in these ecosystems highly relevant for forest management.We therefore assessed the importance of α- and β-diversity of tree species in driving γ-multifunctionality in mature European forests. To do so, we used data taken from a pan-European forest dataset consisting of 209 forest plots, specifically selected to investigate relationships between tree diversity and ecosystem functioning by maximizing variation in dominant “target” species richness and minimizing (i) variation in other potential drivers of ecosystem function (e.g., soil and climatic conditions) and (ii) covariation between tree α-diversity, species composition, and environmental variables as much as possible (38). Our plot selection therefore aimed to mimic biodiversity experiments to investigate relationships between biodiversity and ecosystem functioning in mature forests, which are difficult to undertake with manipulative approaches due to the longevity of tree species. The plots were widely distributed across six European countries, spanning boreal to Mediterranean zones and representing six major European forest types (38). In each plot, 16 ecosystem processes, functions, or properties (termed “functions” hereafter) were measured. These functions represented a wide range of supporting, provisioning, regulating, and cultural ecosystem services (sensu 9) (SI Appendix, Table S3). Next, we created simulated landscapes by randomly drawing plots from a country to generate a “landscape” of five plots, from which γ-multifunctionality was calculated. We then explored relationships between α- and β-diversity and different measures of γ-multifunctionality: threshold-based γ-multifunctionality, quantified as the number of functions with levels above a threshold [a certain percentage of maximum functioning observed across all plots (10)] in at least one plot of the landscape (quantification is shown in Fig. 1B), and sum-based γ-multifunctionality, quantified as the sum of scaled values of all functions across all plots within a landscape (quantification is shown in Fig. 1B). To demonstrate how α- and β-diversity can promote threshold-based γ-multifunctionality, we also measured the relationships between both α- and β-diversity and threshold-based α- and β-multifunctionality (quantification is shown in Fig. 1B).     

Glucose buffer is suitable for blood group conversion with α-N acetylgalactosaminidase and α-galactosidase

Background

It is well known that the buffer plays a key role in the enzymatic reaction involved in blood group conversion. In previous study, we showed that a glycine buffer is suitable for A to O or B to O blood group conversion. In this study, we investigated the use of 5% glucose and other buffers for A to O or B to O blood group conversion by α-N-acetylgalactosaminidase or α-galactosidase.

Materials and methods

We compared the binding ability of α-N-acetylgalactosaminidase/α-galactosidase with red blood cells (RBC) in different reaction buffers, such as normal saline, phosphate-buffered saline (PBS), a disodium hydrogen phosphate-based buffer (PCS), and 5% commercial glucose solution. The doses of enzymes necessary for the A/B to O conversion in different reaction buffers were determined and compared. The enzymes’ ability to bind to RBC was evaluated by western blotting, and routine blood typing and fluorescence activated cell sorting was used to evaluate B/A to O conversion efficiency.

Results

The A to O conversion efficiency in glucose buffer was similar to that in glycine buffer with the same dose (>0.06 mg/mL pRBC). B to O conversion efficiency in glucose buffer was also similar to that in glycine buffer with the same dose (>0.005 mg/mL pRBC). Most enzymes could bind with RBC in glycine or glucose buffer, but few enzymes could bind with RBC in PBS, PCS, or normal saline.

Conclusion

These results indicate that 5% glucose solution provides a suitable condition for enzymolysis, especially for enzymes combining with RBC. Meanwhile, the conversion efficiency of A/B to O was similar in glucose buffer and glycine buffer. Moreover, 5% glucose solution has been used for years in venous transfusion, it is safe for humans and its cost is lower. Our results do, therefore, suggest that 5% glucose solution could become a novel suitable buffer for A/B to O blood group conversion.     

Small heat-shock proteins interact with a flanking domain to suppress polyglutamine aggregation

Small heat-shock proteins (sHsps) are molecular chaperones that play an important protective role against cellular protein misfolding by interacting with partially unfolded proteins on their off-folding pathway, preventing their aggregation. Polyglutamine (polyQ) repeat expansion leads to the formation of fibrillar protein aggregates and neuronal cell death in nine diseases, including Huntington disease and the spinocerebellar ataxias (SCAs). There is evidence that sHsps have a role in suppression of polyQ-induced neurodegeneration; for example, the sHsp alphaB-crystallin (αB-c) has been identified as a suppressor of SCA3 toxicity in a Drosophila model. However, the molecular mechanism for this suppression is unknown. In this study we tested the ability of αB-c to suppress the aggregation of a polyQ protein. We found that αB-c does not inhibit the formation of SDS-insoluble polyQ fibrils. We further tested the effect of αB-c on the aggregation of ataxin-3, a polyQ protein that aggregates via a two-stage aggregation mechanism. The first stage involves association of the N-terminal Josephin domain followed by polyQ-mediated interactions and the formation of SDS-resistant mature fibrils. Our data show that αB-c potently inhibits the first stage of ataxin-3 aggregation; however, the second polyQ-dependent stage can still proceed. By using NMR spectroscopy, we have determined that αB-c interacts with an extensive region on the surface of the Josephin domain. These data provide an example of a domain/region flanking an amyloidogenic sequence that has a critical role in modulating aggregation of a polypeptide and plays a role in the interaction with molecular chaperones to prevent this aggregation.     

Pregnancy without progesterone in horses defines a second endogenous biopotent progesterone receptor agonist, 5α-dihydroprogesterone

One of the most widely accepted axioms of mammalian reproductive biology is that pregnancy requires the (sole) support of progesterone, acting in large measure through nuclear progesterone receptors (PRs) in uterine and cervical tissues, without which pregnancy cannot be established or maintained. However, mares lack detectable progesterone in the latter half of pregnancy. Instead of progesterone, several (mainly 5α-reduced) pregnanes are elevated and have long been speculated to provide progestational support in lieu of progesterone itself. To the authors'' knowledge, evidence for the bioactivity of a second potent endogenously synthesized pregnane able to support pregnancy in the absence of progesterone has never before been reported. The 5α-reduced progesterone metabolite dihydroprogesterone (DHP) was shown in vivo to stimulate endometrial growth and progesterone-dependent gene expression in the horse at subphysiological concentrations and to maintain equine pregnancy in the absence of luteal progesterone in the third and fourth weeks postbreeding. Results of in vitro studies indicate that DHP is an equally potent and efficacious endogenous progestin in the horse but that the PR evolved with increased agonistic potency for DHP at the expense of potency toward progesterone based on comparisons with human PR responses. Sequence analysis and available literature indicate that the enzyme responsible for DHP synthesis, 5α-reductase type 1, also adapted primarily to metabolize progesterone and thereby to serve diverse roles in the physiology of pregnancy in mammals. Our confirmation that endogenously synthesized DHP is a biopotent progestin in the horse ends decades of speculation, explaining how equine pregnancies survive without measurable circulating progesterone in the last 4 to 5 mo of gestation.Since first crystallized almost eight decades ago, progesterone has remained the only endogenous member of the progestin class of steroids defined by its singular ability to maintain pregnancy (1), acting, in large measure, through nuclear progesterone receptors (PRs) in uterine and cervical tissues, without which pregnancy cannot be established or maintained (2). Birth is thought to be triggered by a decrease in systemic progesterone concentrations (withdrawal) (3), even though this is not evident in mares (4), women, or guinea pigs (5), a disparity that limits the utility of other animal models for preterm labor (6). The vast majority of studies have focused on measuring progesterone, with most using immunoassays that necessarily cross-react with multiple pregnanes (7), the bioactivity of which remain uncharacterized. This is reasonable because, in contrast to androgens, estrogens, and corticoids, for which multiple natural biopotent analogs are known, no other endogenous pregnane has ever been shown to substitute for progesterone in pregnancy in any mammal.However, over five decades ago, Short (8) reported that circulating progesterone concentrations in pregnant mares were surprisingly low at <4 ng/mL, as did Holtan et al. (4) subsequently. Indeed, Holtan et al. (9) further confirmed by GC-MS that maternal progesterone concentrations in middle to late equine gestation were <0.5 ng/mL, and were low even in fetal circulation (10). Conversely, 5α-reduced metabolites like 5α-dihydroprogesterone (DHP) were very high in both pregnant mares and their fetuses (9, 10). Some human pregnancies that have extremely low concentrations of progesterone can survive to term also, as in patients who have congenital hypobetalipoproteinemia (11), and progesterone is low or undetectable in plasma of pregnant zebras (12), elephants (13), and the rock hyrax (14). Thus, alternative endogenous progestins have been postulated to exist in species other than horses, but definitive evidence of bioactivity is lacking. For instance, the results of studies investigating the activity of identified circulating pregnanes on equine (15) or human (16) myometrial contractility have not been consistent. Consequently, speculation about alternative progestins has been based mostly on binding assays in tissue extracts (17). However, binding assays alone do not reliably predict bioactivity (18, 19), and, to date, an alternative endogenous progestin capable of sustaining pregnancy in the absence of progesterone has yet to be identified in any mammal.Here, we verify by a unique combination of in vivo and in vitro studies that endogenously synthesized pregnane DHP sustains pregnancy in the absence of detectable progesterone in mares by (i) inducing equine endometrial growth and stimulating expression of progesterone-responsive endometrial genes in vivo; (ii) maintaining equine pregnancy after progesterone withdrawal induced by luteal regression; (iii) activating the equine PR (ePR) in vitro with equal efficacy and potency to progesterone itself; and (iv) doing so at concentrations seen during the luteal phase and early pregnancy, as well as in the second half of equine gestation when progesterone itself is undetectable. Collectively, these data establish DHP as a biopotent progestin in the horse at concentrations seen during gestation. Evidence both in vivo and in vitro demonstrating that an endogenously synthesized pregnane is able to sustain pregnancy by activating the nuclear PR at physiological concentrations has not previously been reported for any species to our knowledge. Evolutionary implications relating to the synthetic enzymes involved, and the classical PR itself, were also explored and are discussed.     

Enforcement of γδ-lineage commitment by the pre–T-cell receptor in precursors with weak γδ-TCR signals

Developing thymocytes bifurcate from a bipotent precursor into αβ- or γδ-lineage T cells. Considering this common origin and the fact that the T-cell receptor (TCR) β-, γ-, and δ-chains simultaneously rearrange at the double negative (DN) stage of development, the possibility exists that a given DN cell can express and transmit signals through both the pre-TCR and γδ-TCR. Here, we tested this scenario by defining the differentiation outcomes and criteria for lineage choice when both TCR-β and γδ-TCR are simultaneously expressed in Rag2^−/− DN cells via retroviral transduction. Our results showed that Rag2^−/− DN cells expressing both TCRs developed along the γδ-lineage, down-regulated CD24 expression, and up-regulated CD73 expression, showed a γδ-biased gene-expression profile, and produced IFN-γ in response to stimulation. However, in the absence of Inhibitor of DNA-binding 3 expression and strong γδ-TCR ligand, γδ-expressing cells showed a lower propensity to differentiate along the γδ-lineage. Importantly, differentiation along the γδ-lineage was restored by pre-TCR coexpression, which induced greater down-regulation of CD24, higher levels of CD73, Nr4a2, and Rgs1, and recovery of functional competence to produce IFN-γ. These results confirm a requirement for a strong γδ-TCR ligand engagement to promote maturation along the γδ T-cell lineage, whereas additional signals from the pre-TCR can serve to enforce a γδ-lineage choice in the case of weaker γδ-TCR signals. Taken together, these findings further cement the view that the cumulative signal strength sensed by developing DN cells serves to dictate its lineage choice.T cells can differentiate along distinct αβ- or γδ-cell lineages, but bifurcate from a common bipotent precursor (1, 2). In mice, the earliest subset of T cells contains CD4⁻ CD8⁻ or double-negative (DN) thymocytes, and this can further be divided into four subgroups (DN1–4) based on the expression of CD25 and CD44 (3, 4). Single-cell progenitor analyses have identified the DN3 stage as the point of T-lineage commitment, and also the final stage at which a DN cell specifies its lineage fate as αβ or γδ (1, 5). The αβ- or γδ-lineage choice decision is governed by several factors. Two competing models have been proposed for this process: the stochastic and instructional models (2). Although evidence exists to support either model, a version of the instructional model posits that the strength of signal transduced by the T-cell receptor (TCR) expressed by the DN3 cell dictates its lineage specification (6, 7).The apparent connection between lineage choice and the TCR expressed by the cell can be severed by manipulations of TCR signal strength. We previously noted that stimulating stronger signals via expression of the ERK/MAPK-induced Inhibitor of DNA-binding 3 (Id3) appears to promote the γδ-lineage fate in developing DN3 cells in the absence of TCR expression (8), suggesting a critical role for Id3 in mediating αβ- versus γδ-lineage decisions at this developmental checkpoint. Nevertheless, absence of Id3 also appears to favor the emergence of innate-like Vγ1.1/Vδ6.3 γδ-TCR–bearing T cells from the thymus over other γδ-TCR subsets (9, 10).Several studies have shown that ligand engagement highly influences the αβ- versus γδ-lineage decision because of its effects on γδ-TCR signal strength (6, 7, 9, 11, 12). γδ-TCR–expressing DN3 cells develop along the αβ-lineage and become CD4⁺ CD8⁺ (double-positive, DP) cells in the absence of ligand engagement (7), whereas provision of the ligand, or the use of antibodies to mimic ligand engagement (11), allows these cells to adopt the γδ-lineage fate, remain DN, and down-regulate expression of CD24. Additional signals, such as those mediated by Notch, can also influence αβ- versus γδ-lineage fate outcomes (1, 13–16). We showed that γδ-TCR–bearing thymocytes adopting the γδ-lineage do not require concurrent signals from Notch to mature past the DN3 stage, whereas their pre-TCR–expressing counterparts are completely dependent upon Notch signaling to facilitate their pre-TCR–dependent differentiation to the DP stage (1, 17).Considering the common origin of αβ- and γδ-lineage cells, it is possible for a bipotent DN3 cell to simultaneously express and transmit signals through a functional pre-TCR and a functional γδ-TCR, especially considering that TCR-β, -γ, and -δ genes complete their rearrangements at the DN3 stage. Additionally, γδ-T cells have been shown to contain TCR-β rearrangements (18) and αβ-lineage cells show evidence of both TCR-γ and -δ rearrangements (19–21). In a previous study looking to address the consequences of simultaneously expressing a TCR-β and γδ-TCR in vivo using transgenic (Tg) mice, the numbers of αβ- and γδ-lineage cells in TCR-β/γδ–expressing cells were both high, and comparable to TCR-β- and γδ-TCR-Tg mice, respectively (22). In this case, however, the TCR chains were expressed earlier than physiological for T-cell development, and premature expression of αβ-TCR transgene can lead to aberrant developmental progression (23, 24).Here, we attempt to definitively answer the question of lineage choice by simultaneously expressing TCR-β and γδ-TCR in Rag2^−/− DN3 cells via retroviral transduction followed by in vitro coculture, including limiting dilution and clonal analyses. We now find that Rag2^−/− DN3 cells expressing both pre-TCR and γδ-TCR mature along the γδ-lineage into functionally competent cells that produce IFN-γ in response to stimulation. However, in the absence of Id3 expression and strong γδ-TCR ligand, γδ-expressing cells show a lower propensity to differentiate along the γδ-lineage, but when expressing both pre- and γδ-TCRs, these cells showed increased γδ-lineage differentiation and recover functional competence to produce IFN-γ, indicating that the pre-TCR can serve to enforce to a γδ-lineage choice in the case of weaker γδ-TCR signals. Taken together, these findings further cement the view that the cumulative signal strength sensed by developing DN cells dictates its lineage choice.