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Before a class II molecule can be loaded with antigenic material and reach the surface to engage CD4+ T cells, its chaperone, the class II-associated invariant chain (Ii), is degraded in a stepwise fashion by proteases in endocytic compartments. We have dissected the role of cathepsin S (CatS) in the trafficking and maturation of class II molecules by combining the use of dendritic cells (DC) from CatS(-/-) mice with a new active site-directed probe for direct visualization of active CatS. Our data demonstrate that CatS is active along the entire endocytic route, and that cleavage of the lysosomal sorting signal of Ii by CatS can occur there in mature DC. Genetic disruption of CatS dramatically reduces the flow of class II molecules to the cell surface. In CatS(-/-) DC, the bulk of major histocompatibility complex (MHC) class II molecules is retained in late endocytic compartments, although paradoxically, surface expression of class II is largely unaffected. The greatly diminished but continuous flow of class II molecules to the cell surface, in conjunction with their long half-life, can account for the latter observation. We conclude that in DC, CatS is a major determinant in the regulation of intracellular trafficking of MHC class II molecules.

Antigen-presenting cells (APC) degrade endocytosed antigens into peptides that are bound and presented to T cells by major histocompatibility complex (MHC) class II molecules. Class II molecules are delivered to endocytic compartments by the class II accessory molecule invariant chain (Ii), which itself must be eliminated to allow peptide binding. The cellular location of Ii degradation, as well as the enzymology of this event, are important in determining the sets of antigenic peptides that will bind to class II molecules. Here, we show that the cysteine protease cathepsin S acts in a concerted fashion with other cysteine and noncysteine proteases to degrade mouse Ii in a stepwise fashion. Inactivation of cysteine proteases results in incomplete degradation of Ii, but the extent to which peptide loading is blocked by such treatment varies widely among MHC class II allelic products. These observations suggest that, first, class II molecules associated with larger Ii remnants can be converted efficiently to class II-peptide complexes and, second, that most class II-associated peptides can still be generated in cells treated with inhibitors of cysteine proteases. Surprisingly, maturation of MHC class II in mice deficient in cathepsin D is unaffected, showing that this major aspartyl protease is not involved in degradation of Ii or in generation of the bulk of antigenic peptides.

Background: BALB/c mice control infection with the obligate intracellular parasite Toxoplasma gondii and develop a latent chronic infection in the brain, as do immunocompetent humans. Interferon-gamma-producing CD8+ T cells provide essential protection against T. gondii infection, but the epitopes recognized have so far remained elusive.Methods: We employed caged major histocompatibility complex molecules to generate approximately 250 H-2L(d) tetramers and to distinguish T. gondii-specific CD8+ T cells in BALB/c mice.Results: We identified 2 T. gondii-specific H-2L(d)-restricted T cell epitopes, one from dense granule protein GRA4 and the other from rhoptry protein ROP7. H-2L(d)/GRA4 reactive T cells from multiple organ sources predominated 2 weeks after infection, while the reactivity of the H-2L(d)/ROP7 T cells peaked 6-8 weeks after infection. BALB/c animals infected with T. gondii mutants defective in establishing a chronic infection showed altered levels of antigen-specific T cells, depending on the T. gondii mutant used.Conclusions: Our results shed light on the identity and the parasite stage-specificity of 2 CD8+ T cell epitopes recognized in the acute and chronic phase of infection with T. gondii. [ABSTRACT FROM AUTHOR]

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) impacts multiple anatomical sites. Whether this is due to the virus itself or is a secondary effect caused by the influx and activation of immune cells is not known. Positron emission tomography (PET) with immunoglobulins can provide insights into which sites and cells are activated in a living animal. Our aim is to use two nanobodies as tools to monitor (1) the distribution of antigen presenting cells (APC) by virtue of their Mafa-DR expression profile, (2) virus-infected cells and viral particles using a nanobody against the SARS-CoV-2 spike protein. Two [ 89 Zr]-labeled nanobodies that target the SARS-CoV-2 spike protein and major histocompatability complex (MHC) class II antigens (Mafa-DR), respectively, are used to monitor their distribution during an experimental SARS-CoV-2 infection in a nonhuman primate model. Scans are obtained before infection and on Day 3 and 10 post infection (pi) in two macaques each. The [ 89 Zr]anti-SARS-CoV-2 spike nanobody localized to SARS-CoV-2-associated lung lesions and the nasal mucosa, while the [ 89 Zr]anti-human leukocyte antigen (HLA)-DR nanobody was predominantly found in non-affected lung tissue after infection. We also detected, pi, upregulation of the Mafa-DR signal, indicative of recruitment of professional APCs, in the superior sagittal sinus. [ 89 Zr]-labeled nanobodies show recruitment of macrophages/monocytes in non-lesional lung tissue in cynomolgus macaques after experimental infection with SARS-CoV-2, as well as accumulation of the spike protein in both lung lesions and the nasal mucosa during infection. These results show the possibility of in vivo monitoring the quality and quantity of immune responses during the initial stages of an infection., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Inflammasome activation results in the cleavage of gasdermin D (GSDMD) by pro-inflammatory caspases. The N-terminal domains (GSDMD NT ) oligomerize and assemble pores penetrating the target membrane. As methods to study pore formation in living cells are insufficient, the order of conformational changes, oligomerization, and membrane insertion remained unclear. We have raised nanobodies (VHHs) against human GSDMD and find that cytosolic expression of VHH GSDMD-1 and VHH GSDMD-2 prevents oligomerization of GSDMD NT and pyroptosis. The nanobody-stabilized GSDMD NT monomers partition into the plasma membrane, suggesting that membrane insertion precedes oligomerization. Inhibition of GSDMD pore formation switches cell death from pyroptosis to apoptosis, likely driven by the enhanced caspase-1 activity required to activate caspase-3. Recombinant antagonistic nanobodies added to the extracellular space prevent pyroptosis and exhibit unexpected therapeutic potential. They may thus be suitable to treat the ever-growing list of diseases caused by activation of (non-) canonical inflammasomes., (© 2024. The Author(s).)

The Class I MHC molecule (MHC-I) HLA-E presents peptides that are derived from the signal sequences, either those of other MHC-I products, or of viral type I membrane glycoproteins. Monoclonal antibodies with proven specificity for HLA-E, and with no cross-reactions with other MHC-I products, have yet to be described. To obtain anti-HLA-E-specific antibodies suitable for a range of applications, we generated monoclonal antibodies against a unique feature of HLA-E: its cytoplasmic tail. We created an immunogen by performing an enzymatically catalyzed transpeptidation reaction to obtain a fusion of the cytoplasmic tail of HLA-E with a nanobody that recognizes murine Class II MHC (MHC-II) products. We obtained a mouse monoclonal antibody that recognizes a 13-residue stretch in the HLA-E cytoplasmic tail. We cloned the genes that encode this antibody in expression vectors to place an LPETG sortase recognition motif at the C-terminus of the heavy and light chains. This arrangement allows the site-specific installation of fluorophores or biotin at these C-termini. The resulting immunoglobulin preparations, labeled with 4 equivalents of a fluorescent or biotinylated payload of choice, can then be used for direct immunofluorescence or detection of the tag by fluorescence or by streptavidin-based methods. We also show that the 13-residue sequence can serve as an epitope tag, independent of the site of its placement within a protein's sequence. The antibody can be used diagnostically to stain for HLA-E on patient tumor samples, it can be used as an antibody-epitope tag for extracellular proteins, and it enables research into the unique role of the cytoplasmic tail of HLA-E., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6's mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH-dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome., Competing Interests: TB, MT, XN, AA, SG, IM, SD, AS, AG, MP, JI, JB, HP, FT No competing interests declared, EB JEB reports personal fees from Scorpion Therapeutics, Reactive therapeutics and Olema Oncology, US HLP serves as an advisor to and owns stock in Cerberus Therapeutics. HLP serves as a consultant to Johnson and Johnson, Immatics Therapeutics, Cue Biopharma, Revela Therapeutics, and Tiba Bio, (© 2023, Bates et al.)

The glycoproteins MICA and MICB are upregulated on the surface of cells undergoing stress, for instance due to (viral) infection or malignant transformation. MICA/B are the ligands for the activating receptor NKG2D, found on cytotoxic immune cells like NK cells, CD8 + T cells, and γδ T cells. Upon engagement of NKG2D, these cells are activated to eradicate the MICA/B-positive targets, assisted by the secretion of cytokines. Nanobodies, or VHHs, are derived from the variable regions of camelid heavy-chain only immunoglobulins. Nanobodies are characterized by their small size, ease of production, stability, and specificity of recognition. We generated nanobodies that recognize membrane-bound MICA with high affinity. Here, we use these nanobodies as building blocks for a chimeric antigen receptor (CAR) to establish VHH-based CAR NK cells. These anti-MICA nanobody-based CAR NK cells recognize and selectively kill MICA-positive tumor cells in vitro and in vivo. We track localization of the VHH-based CAR NK cells to MICA-positive lung metastases by immuno-positron emission tomography imaging., (Published by Oxford University Press on behalf of National Academy of Sciences 2024.)

MICA and MICB are Class I MHC-related glycoproteins that are upregulated on the surface of cells in response to stress, for instance due to infection or malignant transformation. MICA/B are ligands for NKG2D, an activating receptor on NK cells, CD8 + T cells, and γδ T cells. Upon engagement of MICA/B with NKG2D, these cytotoxic cells eradicate MICA/B-positive targets. MICA is frequently overexpressed on the surface of cancer cells of epithelial and hematopoietic origin. Here, we created nanobodies that recognize MICA. Nanobodies, or VHHs, are the recombinantly expressed variable regions of camelid heavy chain-only immunoglobulins. They retain the capacity of antigen recognition but are characterized by their stability and ease of production. The nanobodies described here detect surface-disposed MICA on cancer cells in vitro by flow cytometry and can be used therapeutically as nanobody-drug conjugates when fused to the Maytansine derivative DM1. The nanobody-DM1 conjugate selectively kills MICA positive tumor cells in vitro., Competing Interests: Author KW serves on the scientific advisory boards of DEM BioPharma, Solu Therapeutics, D2M Biotherapeutics and Nextechinvest. He is a co-founder of Immunitas Therapeutics and receives sponsored research funding from Novartis and Fate Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Verhaar, Knoflook, Pishesha, Liu, van Keizerswaard, Wucherpfennig and Ploegh.)

Interferon-induced ubiquitin (Ub)-like modifier ISG15 covalently modifies host and viral proteins to restrict viral infections. Its function is counteracted by the canonical deISGylase USP18 or Ub-specific protease 18. Notwithstanding indications for the existence of other ISG15 cross-reactive proteases, these remain to be identified. Here, we identify deubiquitinase USP16 as an ISG15 cross-reactive protease by means of ISG15 activity-based profiling. Recombinant USP16 cleaved pro-ISG15 and ISG15 isopeptide-linked model substrates in vitro, as well as ISGylated substrates from cell lysates. Moreover, interferon-induced stimulation of ISGylation was increased by depletion of USP16. The USP16-dependent ISG15 interactome indicated that the deISGylating function of USP16 may regulate metabolic pathways. Targeted enzymes include malate dehydrogenase, cytoplasmic superoxide dismutase 1, fructose-bisphosphate aldolase A, and cytoplasmic glutamic-oxaloacetic transaminase 1. USP16 may thus contribute to the regulation of a subset of metabolism-related proteins during type-I interferon responses., Competing Interests: Competing interests statement:The authors declare no competing interest.

The immune system eliminates pathogen intruders such as viruses and bacteria. To recruit immune effectors to virus-infected cells, we conjugated a small molecule, the influenza neuraminidase inhibitor zanamivir, to a nanobody that recognizes the kappa light chains of mouse immunoglobulins. This adduct was designed to achieve half-life extension of zanamivir through complex formation with the much-larger immunoglobulins in the circulation. The zanamivir moiety targets the adduct to virus-infected cells, whereas the anti-kappa component simultaneously delivers polyclonal immunoglobulins of indeterminate specificity and all isotypes. Activation of antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity promoted elimination of influenza neuraminidase-positive cells. A single dose of the conjugate protected mice against influenza A or B viruses and was effective even when given several days after infection with a lethal dose of virus. In the absence of circulating immunoglobulins, we observed no in vivo protection from the adduct. The type of conjugates described here may thus find application for both anti-influenza prophylaxis and therapy.

Invariant natural killer T (iNKT) cells act at the interface between lipid metabolism and immunity because of their restriction to lipid antigens presented on CD1d by antigen-presenting cells (APCs). How foreign lipid antigens are delivered to APCs remains elusive. Since lipoproteins routinely bind glycosylceramides structurally similar to lipid antigens, we hypothesized that circulating lipoproteins form complexes with foreign lipid antigens. In this study, we used 2-color fluorescence correlation spectroscopy to show, for the first time to our knowledge, stable complex formation of lipid antigens α-galactosylceramide (αGalCer), isoglobotrihexosylceramide, and OCH, a sphingosine-truncated analog of αGalCer, with VLDL and/or LDL in vitro and in vivo. We demonstrate LDL receptor-mediated (LDLR-mediated) uptake of lipoprotein-αGalCer complexes by APCs, leading to potent complex-mediated activation of iNKT cells in vitro and in vivo. Finally, LDLR-mutant PBMCs of patients with familial hypercholesterolemia showed impaired activation and proliferation of iNKT cells upon stimulation, underscoring the relevance of lipoproteins as a lipid antigen delivery system in humans. Taken together, circulating lipoproteins form complexes with lipid antigens to facilitate their transport and uptake by APCs, leading to enhanced iNKT cell activation. This study thereby reveals a potentially novel mechanism of lipid antigen delivery to APCs and provides further insight into the immunological capacities of circulating lipoproteins.

Drug platforms that enable the directed delivery of therapeutics to sites of diseases to maximize efficacy and limit off-target effects are needed. Here, we report the development of PROT 3 EcT, a suite of commensal Escherichia coli engineered to secrete proteins directly into their surroundings. These bacteria consist of three modular components: a modified bacterial protein secretion system, the associated regulatable transcriptional activator, and a secreted therapeutic payload. PROT 3 EcT secrete functional single-domain antibodies, nanobodies (Nbs), and stably colonize and maintain an active secretion system within the intestines of mice. Furthermore, a single prophylactic dose of a variant of PROT 3 EcT that secretes a tumor necrosis factor-alpha (TNF-α)-neutralizing Nb is sufficient to ablate pro-inflammatory TNF levels and prevent the development of injury and inflammation in a chemically induced model of colitis. This work lays the foundation for developing PROT 3 EcT as a platform for the treatment of gastrointestinal-based diseases., Competing Interests: Declaration of interests C.F.L is on the scientific advisory board (SAB) for and A.Z.R. is an employee and has equity at Synlogic Therapeutics. J.P.L is an employee at AdAlta Ltd. W.S.G. is on the SABs of Kintai Therapeutics, SanaRx, Evelo Biosciences, and Tenza. F.I.S. is a co-founder and shareholder of Odyssey Therapeutics and a shareholder of IFM Therapeutics. C.B.S is a scientific advisor, and V.K. is the CEO and co-founder of Vicero, Inc. PROT(3)EcT is the subject of US Patent no. 9,951,340 and US Patent 10,702,559, both filed by Massachusetts General Hospital., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Inflammasomes integrate cytosolic evidence of infection or damage to mount inflammatory responses. The inflammasome sensor NLRP1 is expressed in human keratinocytes and coordinates inflammation in the skin. We found that diverse stress signals induce human NLRP1 inflammasome assembly by activating MAP kinase p38: While the ribotoxic stress response to UV and microbial molecules exclusively activates p38 through MAP3K ZAKα, infection with arthropod-borne alphaviruses, including Semliki Forest and Chikungunya virus, activates p38 through ZAKα and potentially other MAP3K. We demonstrate that p38 directly phosphorylates NLRP1 and that serine 107 in the linker region is critical for activation. NLRP1 phosphorylation is followed by ubiquitination of NLRP1PYD, N-terminal degradation of NLRP1, and nucleation of inflammasomes by NLRP1UPA-CARD. In contrast, activation of NLRP1 by nanobody-mediated ubiquitination, viral proteases, or inhibition of DPP9 was independent of p38 activity. Taken together, we define p38 activation as a unifying signaling hub that controls NLRP1 inflammasome activation by integrating a variety of cellular stress signals relevant to the skin., (© 2022 Jenster et al.)

Antigen processing and presentation are the cornerstones of adaptive immunity. B cells cannot generate high-affinity antibodies without T cell help. CD4 + T cells, which provide such help, use antigen-specific receptors that recognize major histocompatibility complex (MHC) molecules in complex with peptide cargo. Similarly, eradication of virus-infected cells often depends on cytotoxic CD8 + T cells, which rely on the recognition of peptide-MHC complexes for their action. The two major classes of glycoproteins entrusted with antigen presentation are the MHC class I and class II molecules, which present antigenic peptides to CD8 + T cells and CD4 + T cells, respectively. This Review describes the essentials of antigen processing and presentation. These pathways are divided into six discrete steps that allow a comparison of the various means by which antigens destined for presentation are acquired and how the source proteins for these antigens are tagged for degradation, destroyed and ultimately displayed as peptides in complex with MHC molecules for T cell recognition., (© 2022. Springer Nature Limited.)

Nanobodies have emerged as powerful protein-binding tools to uncover protein functions. Using functionalized protein binders, proteins of interest can be visualized, degraded, delocalized, or post-translationally modified in vivo. We recently reported the use of two short peptide tags, 10-aa 127D01 and 14-aa VHH05, and their corresponding nanobodies, Nb127D01 and NbVHH05, for both in vitro and in vivo studies in Drosophila. Here, we provide detailed protocols for nanobody production and for visualization of proteins of interest in either fixed or live samples. In addition, we include protocols for endogenous protein tagging using CRISPR-mediated genome engineering. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Nanobody production in S2 cells Basic Protocol 2: Nanobody expression and purification in bacterial cells Basic Protocol 3: Immunostaining with nanobodies Basic Protocol 4: Immunoblotting with nanobodies Basic Protocol 5: Immunoprecipitation with nanobodies prepared from S2 cells Basic Protocol 6: Immunoprecipitation with nanobodies prepared from bacteria Basic Protocol 7: NbVHH05 and Nb127D01 used as chromobodies Basic Protocol 8: NanoTag trap as a method to alter protein localization Support Protocol: CRISPR-mediated tagging of endogenous genes with NanoTags., (© 2022 Wiley Periodicals LLC.)

The distribution of Ly6C/G-positive cells in response to an infection of the mouse respiratory tract with influenza A virus was followed noninvasively over time by immuno-positron emission tomography. We converted nanobodies that recognize Ly6C and Ly6G, markers of neutrophils and other myeloid cells, as well as an influenza hemagglutinin-specific nanobody, into 89 Zr-labeled PEGylated positron emission tomography (PET) imaging agents. The PET images showed strong accumulation of these imaging agents in the lungs of infected mice. Immunohistochemistry of influenza virus-infected mice and control mice, injected with a biotinylated and PEGylated version of the Ly6C/G-specific nanobody, showed the presence of abundant Ly6C/G-positive myeloid cells and positivity for Ly6C/G on bronchial epithelium in influenza virus-infected mice. This is consistent with focal inflammation in the lungs, a finding that correlated well with the immuno-PET results. No such signals were detected in control mice. Having shown by PET the accumulation of the Ly6C/G-specific nanobody in infected lungs, we synthesized conjugates of Ly6C/G-specific nanobodies with dexamethasone to enable targeted delivery of this immunosuppressive corticosteroid to sites of inflammation. Such conjugates reduced the weight loss that accompanies infection, while the equivalent amount of free dexamethasone was without effect. Nanobody-drug conjugates thus enable delivery of drugs to particular cell types at the appropriate anatomic site(s). By avoiding systemic exposure to free dexamethasone, this strategy minimizes its undesirable side effects because of the much lower effective dose of the nanobody-dexamethasone conjugate. The ability to selectively target inflammatory cells may find application in the treatment of other infections or other immune-mediated diseases.

The microbiome contributes to the development and maturation of the immune system. In response to commensal bacteria, intestinal CD4 + T lymphocytes differentiate into functional subtypes with regulatory or effector functions. The development of small intestine intraepithelial lymphocytes that coexpress CD4 and CD8αα homodimers (CD4IELs) depends on the microbiota. However, the identity of the microbial antigens recognized by CD4 + T cells that can differentiate into CD4IELs remains unknown. We identified β-hexosaminidase, a conserved enzyme across commensals of the Bacteroidetes phylum, as a driver of CD4IEL differentiation. In a mouse model of colitis, β-hexosaminidase-specific lymphocytes protected against intestinal inflammation. Thus, T cells of a single specificity can recognize a variety of abundant commensals and elicit a regulatory immune response at the intestinal mucosa.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development., Competing Interests: Declaration of interests F.B., E.N., T.R.O., I.Z., D.J.D., and O.L. are named inventors on invention disclosures and patents involving vaccine adjuvants. S.J.E. is a founder of TSCAN Therapeutics, ImmuneID, MAZE Therapeutics, and Mirimus. S.J.E. serves on the scientific advisory board of Homology Medicines, TSCAN Therapeutics, MAZE Therapetics, and XChem, and is an advisor for MPM, none of which impact this work. S.J.E. is an inventor on a patent application issued to the Brigham and Women’s Hospital (US20160320406A) that covers the use of the VirScan library to identify pathogen antibodies in blood. The other authors declare no commercial or financial conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Expansion of the available repertoire of reagents for visualization and manipulation of proteins will help understand their function. Short epitope tags linked to proteins of interest and recognized by existing binders such as nanobodies facilitate protein studies by obviating the need to isolate new antibodies directed against them. Nanobodies have several advantages over conventional antibodies, as they can be expressed and used as tools for visualization and manipulation of proteins in vivo. Here, we characterize two short (<15aa) NanoTag epitopes, 127D01 and VHH05, and their corresponding high-affinity nanobodies. We demonstrate their use in Drosophila for in vivo protein detection and re-localization, direct and indirect immunofluorescence, immunoblotting, and immunoprecipitation. We further show that CRISPR-mediated gene targeting provides a straightforward approach to tagging endogenous proteins with the NanoTags. Single copies of the NanoTags, regardless of their location, suffice for detection. This versatile and validated toolbox of tags and nanobodies will serve as a resource for a wide array of applications, including functional studies in Drosophila and beyond., Competing Interests: JX, AK, RC, FF, JL, YF, ES, RB, SM, JZ, HP, NP No competing interests declared, (© 2022, Xu et al.)

Low plasma levels of Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) are associated with decreased low-density lipoprotein (LDL) cholesterol and a reduced risk of cardiovascular disease. PCSK9 binds to the epidermal growth factor-like repeat A (EGFA) domain of LDL receptors (LDLR), very low-density lipoprotein receptors (VLDLR), apolipoprotein E receptor 2 (ApoER2), and lipoprotein receptor-related protein 1 (LRP1) and accelerates their degradation, thus acting as a key regulator of lipid metabolism. Antibody and RNAi-based PCSK9 inhibitor treatments lower cholesterol and prevent cardiovascular incidents in patients, but their high-cost hampers market penetration. We sought to develop a safe, long-term and one-time solution to treat hyperlipidemia. We created a cDNA encoding a chimeric protein in which the extracellular N- terminus of red blood cells (RBCs) specific glycophorin A was fused to the LDLR EGFA domain and introduced this gene into mouse bone marrow hematopoietic stem and progenitor cells (HSPCs). Following transplantation into irradiated mice, the animals produced RBCs with the EGFA domain (EGFA-GPA RBCs) displayed on their surface. These animals showed significantly reduced plasma PCSK9 (66.5% decrease) and reduced LDL levels (40% decrease) for as long as 12 months post-transplantation. Furthermore, the EGFA- GPA mice remained lean for life and maintained normal body weight under a high-fat diet. Hematopoietic stem cell gene therapy can generate red blood cells expressing an EGFA-glycophorin A chimeric protein as a practical and long-term strategy for treating chronic hyperlipidemia and obesity., Competing Interests: H.F.L. and H.L.P. served as scientific advisors and have/had equity in Rubius, a biotechnology company that seeks to exploit engineered red blood cells as therapeutics but does not provide financial support for the technology described in this paper. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Immuno-positron emission tomography (immuno-PET) is a noninvasive imaging method that enables tracking of immune cells in living animals. We used a nanobody that recognizes mouse CD8α and labeled it with 89 Zr to image mouse CD8 + T cells in the course of an infection with influenza A virus (IAV). The CD8 + signal showed a strong increase in the mediastinal lymph node (MLN) and thymus as early as 4 days post-infection (dpi), and as early as 6 dpi in the lungs. Over the course of the infection, CD8 + T cells were at first distributed diffusely throughout the lungs and then accumulated more selectively in specific regions of the lungs. These distributions correlated with morbidity as mice reached the peak of weight loss over this interval. CD8 + T cells obtained from control or IAV-infected mice showed a difference in their distribution and migration when comparing their fate upon labeling ex vivo with 89 Zr-labeled anti-CD8α nanobody and transfer into infected versus control animals. CD8 + T cells from infected mice, upon transfer, appear to be trained to persist in the lungs, even of uninfected mice. Immuno-PET imaging thus allows noninvasive, dynamic monitoring of the immune response to infectious agents in living animals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rothlauf, Li, Pishesha, Xie, Woodham, Bousbaine, Kolifrath, Verschoor and Ploegh.)

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (Spike RBD ) fused to an alpaca-derived nanobody that recognizes class II major histocompatibility complex antigens (VHH MHCII ). This vaccine elicits robust humoral and cellular immunity against SARS-CoV-2 and its variants. Both young and aged mice immunized with two doses of VHH MHCII -Spike RBD elicit high-titer binding and neutralizing antibodies. Immunization also induces strong cellular immunity, including a robust CD8 T cell response. VHH MHCII -Spike RBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy., Competing Interests: Competing interest statement: N.P., T.J.H., and H.L.P. have filed a patent with the Boston Children’s Hospital for the use of nanobody conjugates as immunomodulatory vaccines. P.W.R. and S.P.J.W. have filed a patent with Washington University for VSV-SARS-CoV-2 and its variants. S.P.J.W has received unrelated funding support in sponsored research agreements with Vir Biotechnology, AbbVie, and sAB therapeutics., (Copyright © 2021 the Author(s). Published by PNAS.)

The association of autoimmune diseases with particular allellic products of the class-II major histocompatibility complex (MHCII) region implicates the presentation of the offending self-antigens to T cells. Because antigen-presenting cells are tolerogenic when they encounter an antigen under non-inflammatory conditions, the manipulation of antigen presentation may induce antigen-specific tolerance. Here, we show that, in mouse models of experimental autoimmune encephalomyelitis, type 1 diabetes and rheumatoid arthritis, the systemic administration of a single dose of nanobodies that recognize MHCII molecules and conjugated to the relevant self-antigen under non-inflammatory conditions confers long-lasting protection against these diseases. Moreover, co-administration of a nanobody-antigen adduct and the glucocorticoid dexamethasone, conjugated to the nanobody via a cleavable linker, halted the progression of established experimental autoimmune encephalomyelitis in symptomatic mice and alleviated their symptoms. This approach may represent a means of treating autoimmune conditions., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Antibodies raised against many cell surface proteins, including G protein-coupled receptors, remain important tools for their functional characterization. By linking antibodies to ligands for cell surface proteins, such adducts can be targeted to the surface of a cell type of choice. Site-specific functionalization of full-size antibodies with synthetic moieties remains challenging. Here we present new approaches in which single domain antibodies (known as VHHs or nanobodies) that target either cell surface proteins or conventional antibodies are used to indirectly deliver ligands for GPCRs to their sites of action. The combination of high yield production of nanobodies, facile site-specific functionalization, and compatibility with commercially available mouse and rabbit antibodies should enable wide application of this approach., Competing Interests: R. W. C., T. J. G., and H. L. P. have filed a patent on the use of nanobodies to target GPCRs for signaling (US application number 16810783)., (This journal is © The Royal Society of Chemistry.)

Immuno-positron emission tomography (PET), a noninvasive imaging modality, can provide a dynamic approach for longitudinal assessment of cell populations of interest. Transformation of mAbs into single-chain variable fragment (scFv)-based PET imaging agents would allow noninvasive tracking in vivo of a wide range of possible targets. We used sortase-mediated enzymatic labeling in combination with PEGylation to develop an anti-mouse CD4 scFv-based PET imaging agent constructed from an anti-mouse CD4 mAb. This anti-CD4 scFv can monitor the in vivo distribution of CD4 + T cells by immuno-PET. We tracked CD4 + and CD8 + T cells in wild-type mice, in immunodeficient recipients reconstituted with monoclonal populations of OT-II and OT-I T cells, and in a B16 melanoma model. Anti-CD4 and -CD8 immuno-PET showed that the persistence of both CD4 + and CD8 + T cells transferred into immunodeficient mice improved when recipients were immunized with OVA in CFA. In tumor-bearing animals, infiltration of both CD4 + and CD8 + T cells increased as the tumor grew. The approach described in this study should be readily applicable to convert clinically useful Abs into the corresponding scFv PET imaging agents., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Red blood cells (RBCs) can serve as vascular carriers for drugs, proteins, peptides, and nanoparticles. Human RBCs remain in the circulation for ∼120 days, are biocompatible, and are immunologically largely inert. RBCs are cleared by the reticuloendothelial system and can induce immune tolerance to foreign components attached to the RBC surface. RBC conjugates have been pursued in clinical trials to treat cancers and autoimmune diseases and to correct genetic disorders. Still, most methods used to modify RBCs require multiple steps, are resource-intensive and time-consuming, and increase the risk of inflicting damage to the RBCs. Here, we describe direct conjugation of peptides and proteins onto the surface of RBCs in a single step, catalyzed by a highly efficient, recombinant asparaginyl ligase under mild, physiological conditions. In mice, the modified RBCs remain intact in the circulation, display a normal circulatory half-life, and retain their immune tolerance-inducing properties, as shown for protection against an accelerated model for type 1 diabetes. We conjugated different nanobodies to RBCs with retention of their binding properties, and these modified RBCs can target cancer cells in vitro . This approach provides an appealing alternative to current methods of RBC engineering. It provides ready access to more complex RBC constructs and highlights the general utility of asparaginyl ligases for the modification of native cell surfaces.

While various GPCRs, including US28, display constitutive, ligand-independent activity, it remains to be established whether ligand-dependent and -independent active conformations differ and can be selectively modulated. Previously, the agonist-bound conformation of US28 was stabilized and its structure was solved using the anti-US28 nanobody Nb7. Here we report the recognition of the constitutively active, apo-conformation of US28 by another nanobody VUN103. While the Nb7 intrabody selectively inhibits ligand-induced signaling, the VUN103 intrabody blocks constitutive signaling, indicating the existence of distinct US28 conformational states. By displacing Gα q protein, VUN103 prevents US28 signaling and reduces tumor spheroids growth. Overall, nanobodies specific for distinct GPCR conformational states, i.e. apo- and agonist-bound, can selectively target and discern functional consequences of ligand-dependent versus independent signaling., (© 2021. The Author(s).)

Derlin family members (Derlins) are primarily known as components of the endoplasmic reticulum-associated degradation pathway that eliminates misfolded proteins. Here we report a function of Derlins in the brain development. Deletion of Derlin-1 or Derlin-2 in the central nervous system of mice impaired postnatal brain development, particularly of the cerebellum and striatum, and induced motor control deficits. Derlin-1 or Derlin-2 deficiency reduced neurite outgrowth in vitro and in vivo and surprisingly also inhibited sterol regulatory element binding protein 2 (SREBP-2)-mediated brain cholesterol biosynthesis. In addition, reduced neurite outgrowth due to Derlin-1 deficiency was rescued by SREBP-2 pathway activation. Overall, our findings demonstrate that Derlins sustain brain cholesterol biosynthesis, which is essential for appropriate postnatal brain development and function., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Natural killer (NK) cells are innate cytolytic effectors that target HIV-infected CD4 + T cells. In conjunction with antibodies recognizing the HIV envelope, NK cells also eliminate HIV-infected targets through antibody-dependent cellular cytotoxicity (ADCC). However, how these NK cell functions impact infected macrophages is less understood. We show that HIV-infected macrophages resist NK cell-mediated killing. Compared with HIV-infected CD4 + T cells, initial innate NK cell interactions with HIV-infected macrophages skew the response toward cytokine production, rather than release of cytolytic contents, causing inefficient elimination of infected macrophages. Studies with chimeric antigen receptor (CAR) T cells demonstrate that the viral envelope is equally accessible on CD4 + T cells and macrophages. Nonetheless, ADCC against macrophages is muted compared with ADCC against CD4 + T cells. Thus, HIV-infected macrophages employ mechanisms to evade immediate cytolytic NK cell function while preserving inflammatory cytokine responses. These findings emphasize the importance of eliminating infected macrophages for HIV cure efforts., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In vivo imaging has become in recent years an incredible tool to study biological events and has found critical applications in diagnostic medicine. Although a lot of efforts and applications have been achieved using monoclonal antibodies, other types of delivery agents are being developed. Among them, VHHs, antigen binding fragments derived from camelid heavy chain-only antibodies, also known as nanobodies, have particularly attracted attention. Indeed, their stability, fast clearance, good tissue penetration, high solubility, simple cloning and recombinant production make them attractive targeting agents for imaging modalities such as PET, SPECT or Infra-Red. In this review, we discuss the pioneering work that has been carried out using VHHs and summarize the recent developments that have been made using nanobodies for in vivo , non-invasive, imaging., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

For treatment and diagnosis of cancer, antibodies have proven their value and now serve as a first line of therapy for certain cancers. A unique class of antibody fragments called nanobodies, derived from camelid heavy chain-only antibodies, are gaining increasing acceptance as diagnostic tools and are considered also as building blocks for chimeric antigen receptors as well as for targeted drug delivery. The small size of nanobodies (∼15 kDa), their stability, ease of manufacture and modification for diverse formats, short circulatory half-life, and high tissue penetration, coupled with excellent specificity and affinity, account for their attractiveness. Here we review applications of nanobodies in the sphere of tumor biology., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Nuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 or inner ring complex. Working in S. cerevisiae, and to study the assembly of these two essential subcomplexes, we here develop a set of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. These nanobodies all bind specifically and with high affinity. We present structures of several nup-nanobody complexes, revealing their binding sites. Additionally, constitutive expression of the nanobody suite in S. cerevisiae detect accessible and obstructed surfaces of the Y complex and Nic96 within the NPC. Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.

The adaptive immune system has evolved to recognize with incredible precision a large diversity of molecules. Innovations in high-throughput sequencing and bioinformatics have accelerated large-scale immune repertoire analyses and given us important insights into the behavior of the adaptive immune system. However, establishing a connection between receptor sequence and its antigen-specificity remains a challenge despite its central role in determining T and B cell fate. We discuss recent large-scale antigen discovery technologies which can be combined with adaptive immune receptor repertoire (AIRR) studies. We highlight important discoveries made using repertoire analyses in the field of host-microbe interactions., Competing Interests: Declaration of competing interest The authors declare no competing interests.

Reagents that bind tightly and specifically to biomolecules of interest remain essential in the exploration of biology and in their ultimate application to medicine. Besides ligands for receptors of known specificity, agents commonly used for this purpose are monoclonal antibodies derived from mice, rabbits, and other animals. However, such antibodies can be expensive to produce, challenging to engineer, and are not necessarily stable in the context of the cellular cytoplasm, a reducing environment. Heavy chain-only antibodies, discovered in camelids, have been truncated to yield single-domain antibody fragments (VHHs or nanobodies) that overcome many of these shortcomings. Whereas they are known as crystallization chaperones for membrane proteins or as simple alternatives to conventional antibodies, nanobodies have been applied in settings where the use of standard antibodies or their derivatives would be impractical or impossible. We review recent examples in which the unique properties of nanobodies have been combined with complementary methods, such as chemical functionalization, to provide tools with unique and useful properties., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Cheloha et al.)

Trained immunity, a functional state of myeloid cells, has been proposed as a compelling immune-oncological target. Its efficient induction requires direct engagement of myeloid progenitors in the bone marrow. For this purpose, we developed a bone marrow-avid nanobiologic platform designed specifically to induce trained immunity. We established the potent anti-tumor capabilities of our lead candidate MTP 10 -HDL in a B16F10 mouse melanoma model. These anti-tumor effects result from trained immunity-induced myelopoiesis caused by epigenetic rewiring of multipotent progenitors in the bone marrow, which overcomes the immunosuppressive tumor microenvironment. Furthermore, MTP 10 -HDL nanotherapy potentiates checkpoint inhibition in this melanoma model refractory to anti-PD-1 and anti-CTLA-4 therapy. Finally, we determined MTP 10 -HDL's favorable biodistribution and safety profile in non-human primates. In conclusion, we show that rationally designed nanobiologics can promote trained immunity and elicit a durable anti-tumor response either as a monotherapy or in combination with checkpoint inhibitor drugs., Competing Interests: Declaration of Interests W.J.M.M., L.A.B.J., J.O., Z.A.F., and M.G.N. are scientific co-founders of and have equity in Trained Therapeutix Discovery. W.J.M.M. and Z.A.F. have consulting agreements with Trained Therapeutix Discovery., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Single-stranded, positive-sense RNA viruses assemble their replication complexes in infected cells from a multidomain replication polyprotein. This polyprotein usually contains at least one protease, the primary function of which is to process the polyprotein into mature proteins. Such proteases also may have other functions in the replication cycle. For instance, cysteine proteases (PRO) frequently double up as ubiquitin hydrolases (DUB), thus interfering with cellular processes critical for virus replication. We previously reported the crystal structures of such a PRO/DUB from Turnip yellow mosaic virus ( TYMV ) and of its complex with one of its PRO substrates. Here we report the crystal structure of TYMV PRO/DUB in complex with ubiquitin. We find that PRO/DUB recognizes ubiquitin in an unorthodox way: It interacts with the body of ubiquitin through a split recognition motif engaging both the major and the secondary recognition patches of ubiquitin (Ile 44 patch and Ile 36 patch, respectively, including Leu 8 , which is part of the two patches). However, the contacts are suboptimal on both sides. Introducing a single-point mutation in TYMV PRO/DUB aimed at improving ubiquitin-binding led to a much more active DUB. Comparison with other PRO/DUBs from other viral families, particularly coronaviruses, suggests that low DUB activities of viral PRO/DUBs may generally be fine-tuned features of interaction with host factors., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Fieulaine et al.)

The immune system's ability to recognize peptides on major histocompatibility molecules contributes to the eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate the efficacy of immune interventions and improve mechanistic understanding of immune responses. For this purpose, we employ synTacs, which are dimeric major histocompatibility molecule scaffolds of defined composition. SynTacs, when labeled with positron-emitting isotopes, can noninvasively image antigen-specific CD8 + T cells in vivo. Using radiolabeled synTacs loaded with the appropriate peptides, we imaged human papillomavirus-specific CD8 + T cells by positron emission tomography in mice bearing human papillomavirus-positive tumors, as well as influenza A virus-specific CD8 + T cells in the lungs of influenza A virus-infected mice. It is thus possible to visualize antigen-specific CD8 + T-cell populations in vivo, which may serve prognostic and diagnostic roles.