Expression of tdTomato and luciferase in a murine lung cancer alters the growth and immune microenvironment of the tumor.

Imaging techniques based on fluorescence and bioluminescence have been important tools in visualizing tumor progression and studying the effect of drugs and immunotherapies on tumor immune microenvironment in animal models of cancer. However, transgenic expression of foreign proteins may induce immune responses in immunocompetent syngeneic tumor transplant models and augment the efficacy of experimental drugs. In this study, we show that the growth rate of Lewis lung carcinoma (LL/2) tumors was reduced after transduction of tdTomato and luciferase (tdTomato/Luc) compared to the parental cell line. tdTomato/Luc expression by LL/2 cells altered the tumor microenvironment by increasing tumor-infiltrating lymphocytes (TILs) while inhibiting tumor-induced myeloid-derived suppressor cells (MDSCs). Interestingly, tdTomato/Luc expression did not alter the response of LL/2 tumors to anti-PD-1 and anti-CTLA-4 antibodies. These results suggest that the use of tdTomato/Luc-transduced cancer cells to conduct studies in immune competent mice may lead to cell-extrinsic tdTomato/Luc-induced alterations in tumor growth and tumor immune microenvironment that need to be taken into consideration when evaluating the efficacy of anti-cancer drugs and vaccines in immunocompetent animal models.

Disruption of Cxcr3 chemotactic signaling alters lysosomal function and renders macrophages more microbicidal.

Chemotaxis and lysosomal function are closely intertwined processes essential for the inflammatory response and clearance of intracellular bacteria. We used the zebrafish model to examine the link between chemotactic signaling and lysosome physiology in macrophages during mycobacterial infection and wound-induced inflammation in vivo. Macrophages from zebrafish larvae carrying a mutation in a chemokine receptor of the Cxcr3 family display upregulated expression of vesicle trafficking and lysosomal genes and possess enlarged lysosomes that enhance intracellular bacterial clearance. This increased microbicidal capacity is phenocopied by inhibiting the lysosomal transcription factor EC, while its overexpression counteracts the protective effect of chemokine receptor mutation. Tracking macrophage migration in zebrafish revealed that lysosomes of chemokine receptor mutants accumulate in the front half of cells, preventing macrophage polarization during chemotaxis and reaching sites of inflammation. Our work shows that chemotactic signaling affects the bactericidal properties and localization during chemotaxis, key aspects of the inflammatory response.

Generation of mWasabi fluorescent protein-binding nanobodies.

mWasabi is a bright monomeric green fluorescent protein. It can be used as a fusion tag to monitor various biological events, e.g. protein localization. Here we report the selection of camelid-derived single-domain antibody fragments (nanobodies) against mWasabi. In this work, phage-display approach was employed to select the high affinity mWasabi-specific Nb (nanobodies). These nanobodies were able to recognize mWasabi or in a fused fashion with PD1. The interesting binding characteristics of these two mWasabi-specific nanobodies could be valuable for design new tools for cellular tracing or targeting based on the mWasabi-fusing protein in many different biological research fields.

Quantitative immunohistochemistry using an antibody-fused bioluminescent protein.

We established a quantitative detection method for immunohistochemistry based on a reference standard light-emitting diode, protein microarray and antibody-fused bioluminescent protein. In this procedure, we calibrated the bioluminescence imaging system and prepared the calibration curve between antigen and antibody-fused bioluminescent protein using a protein microarray. Then we converted the detecting light signal to antigen count via absolute photon number in the bioluminescent images; there was a resulting threefold difference in the target antigen number between normal and cancerous tissues. Our technique can easily compare immunohistological images and evaluate tumor progression in quantitative pathological diagnosis.

Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors.

DNA point accumulation for imaging in nanoscale topography (PAINT) is a rapidly developing fluorescence super-resolution technique, which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20⁻25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using three nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the three targets with 20 nm resolution, and within only 35 minutes acquisition time.

Ratiometric Bioluminescent Sensor Proteins Based on Intramolecular Split Luciferase Complementation.

Bioluminescent sensor proteins provide attractive tools for applications ranging from in vivo imaging to point-of-care testing. Here we introduce a new class of ratiometric bioluminescent sensor proteins that do not rely on direct modulation of BRET efficiency, but are based on competitive intramolecular complementation of split NanoLuc luciferase. Proof of concept for the feasibility of this sensor principle was provided by developing a blue-red light emitting sensor protein for the detection of anti-HIV1-p17 antibodies with a 500% change in emission ratio and a Kd of 10 pM. The new sensor design also improved the dynamic response of a sensor for the therapeutic antibody cetuximab 4-fold, allowing the direct quantification of this anti-EGFR antibody in undiluted blood plasma. The modular sensor architecture allows easy and systematic tuning of a sensor's dynamic range and should be generally applicable to allow rational engineering of bioluminescent sensor proteins.

The mKate fluorescent protein expressed by Leishmania mexicana modifies the parasite immunopathogenicity in BALB/c mice.

Parasites have been engineered to express fluorescent reporter proteins, yet the impact of red fluorescent proteins on Leishmania infections remains largely unknown. We analysed the infection outcome of Leishmania mexicana parasites engineered for the constitutive expression of mKate protein and evaluated their immunogenicity in BALB/c mice. Infection of BALB/c mice with mKate transfected L. mexicana (LmexmKate ) parasites caused enlarged lesion sizes, leading to ulceration, and containing more parasites, as compared to LmexWT . The mKate protein showed immunogenic properties inducing antibody production against the mKate protein, as well as enhancing antibody production against the parasite. The augmented lesion sizes and ulcers, together with the more elevated antibody production, were related to an enhanced number of TNF-α and IL-1ß producing cells in the infected tissues. We conclude that mKate red fluorescent protein is an immunogenic protein, capable of modifying disease evolution of L. mexicana.

Competitive repopulation of an empty microglial niche yields functionally distinct subsets of microglia-like cells.

Circulating monocytes can compete for virtually any tissue macrophage niche and become long-lived replacements that are phenotypically indistinguishable from their embryonic counterparts. As the factors regulating this process are incompletely understood, we studied niche competition in the brain by depleting microglia with >95% efficiency using Cx3cr1CreER/+R26DTA/+ mice and monitored long-term repopulation. Here we show that the microglial niche is repopulated within weeks by a combination of local proliferation of CX3CR1+F4/80lowClec12a- microglia and infiltration of CX3CR1+F4/80hiClec12a+ macrophages that arise directly from Ly6Chi monocytes. This colonization is independent of blood brain barrier breakdown, paralleled by vascular activation, and regulated by type I interferon. Ly6Chi monocytes upregulate microglia gene expression and adopt microglia DNA methylation signatures, but retain a distinct gene signature from proliferating microglia, displaying altered surface marker expression, phagocytic capacity and cytokine production. Our results demonstrate that monocytes are imprinted by the CNS microenvironment but remain transcriptionally, epigenetically and functionally distinct.

GPR108, an NF-κB activator suppressed by TIRAP, negatively regulates TLR-triggered immune responses.

Higher vertebrates have evolved innate and adaptive immune systems to defend against foreign substances and pathogens. Sophisticated regulatory circuits are needed to avoid inappropriate immune responses and inflammation. GPR108 is a seven-transmembrane family protein that activates NF-κB strongly when overexpressed. Surprisingly, its action in a physiological context is that of an antagonist of Toll-like receptor (TLR)-mediated signaling. Cells from Gpr108-null mice exhibit enhanced cytokine secretion and NF-κB and IRF3 signaling, whereas Gpr108-null macrophages reconstituted with GPR108 exhibit blunted signaling. Co-expression of TLRs and GPR108 reduces NF-κB and IFNß promoter activation compared to expression of either TLRs or GPR108 alone. Upon TLR stimulation GPR108 abundance increases and the protein engages TLRs and their partners to reduce MyD88 expression and interfere with its binding to TLR4 through blocking MyD88 ubiquitination. In turn GPR108 is antagonized by TIRAP, an adaptor protein for TLR and MyD88. The interrelationships between GPR108 and innate immune signaling components are multifactorial and point to a membrane-associated signaling structure of significant complexity.

Bioluminescent Antibodies for Point-of-Care Diagnostics.

We introduce a general method to transform antibodies into ratiometric, bioluminescent sensor proteins for the no-wash quantification of analytes. Our approach is based on the genetic fusion of antibody fragments to NanoLuc luciferase and SNAP-tag, the latter being labeled with a synthetic fluorescent competitor of the antigen. Binding of the antigen, here synthetic drugs, by the sensor displaces the tethered fluorescent competitor from the antibody and disrupts bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. The semisynthetic sensors display a tunable response range (submicromolar to submillimolar) and large dynamic range (ΔRmax >500 %), and they permit the quantification of analytes through spotting of the samples onto paper followed by analysis with a digital camera.

In Vivo Visualization of Tumor Antigen-containing Microparticles Generated in Fluorescent-protein-elicited Immunity.

In vivo optical spatio-temporal imaging of the tumor microenvironment is useful to explain how tumor immunotherapies work. However, the lack of fluorescent antigens with strong immunogenicity makes it difficult to study the dynamics of how tumors are eliminated by any given immune response. Here, we develop an effective fluorescent model antigen based on the tetrameric far-red fluorescent protein KatushkaS158A (tfRFP), which elicits both humoral and cellular immunity. We use this fluorescent antigen to visualize the dynamic behavior of immunocytes as they attack and selectively eliminate tfRFP-expressing tumors in vivo; swarms of immunocytes rush toward tumors with high motility, clusters of immunocytes form quickly, and numerous antigen-antibody complexes in the form of tfRFP(+) microparticles are generated in the tumor areas and ingested by macrophages in the tumor microenvironment. Therefore, tfRFP, as both a model antigen and fluorescent reporter, is a useful tool to visualize specific immune responses in vivo.

Cellular GFP Toxicity and Immunogenicity: Potential Confounders in in Vivo Cell Tracking Experiments.

Green Fluorescent protein (GFP), used as a cellular tag, provides researchers with a valuable method of measuring gene expression and cell tracking. However, there is evidence to suggest that the immunogenicity and cytotoxicity of GFP potentially confounds the interpretation of in vivo experimental data. Studies have shown that GFP expression can deteriorate over time as GFP tagged cells are prone to death. Therefore, the cells that were originally marked with GFP do not survive and cannot be accurately traced over time. This review will present current evidence for the immunogenicity and cytotoxicity of GFP in in vivo studies by characterizing these responses.

Signal transduction controls heterogeneous NF-κB dynamics and target gene expression through cytokine-specific refractory states.

Cells respond dynamically to pulsatile cytokine stimulation. Here we report that single, or well-spaced pulses of TNFα (>100 min apart) give a high probability of NF-κB activation. However, fewer cells respond to shorter pulse intervals (<100 min) suggesting a heterogeneous refractory state. This refractory state is established in the signal transduction network downstream of TNFR and upstream of IKK, and depends on the level of the NF-κB system negative feedback protein A20. If a second pulse within the refractory phase is IL-1ß instead of TNFα, all of the cells respond. This suggests a mechanism by which two cytokines can synergistically activate an inflammatory response. Gene expression analyses show strong correlation between the cellular dynamic response and NF-κB-dependent target gene activation. These data suggest that refractory states in the NF-κB system constitute an inherent design motif of the inflammatory response and we suggest that this may avoid harmful homogenous cellular activation.

Optimizing selection of large animals for antibody production by screening immune response to standard vaccines.

Antibodies made in large animals are integral to many biomedical research endeavors. Domesticated herd animals like goats, sheep, donkeys, horses and camelids all offer distinct advantages in antibody production. However, their cost of use is often prohibitive, especially where poor antigen response is commonplace; choosing a non-responsive animal can set a research program back or even prevent experiments from moving forward entirely. Over the course of production of antibodies from llamas, we found that some animals consistently produced a higher humoral antibody response than others, even to highly divergent antigens, as well as to their standard vaccines. Based on our initial data, we propose that these "high level responders" could be pre-selected by checking antibody titers against common vaccines given to domestic farm animals. Thus, time and money can be saved by reducing the chances of getting poor responding animals and minimizing the use of superfluous animals.

Interleukin-12-producing CD103+ CD11b- CD8+ dendritic cells are responsible for eliciting gut intraepithelial lymphocyte response against Encephalitozoon cuniculi.

Microsporidia, which belong to the kingdom Fungi, are important opportunistic pathogens in HIV-infected populations and organ transplant recipients that are often associated with a broad range of symptoms, such as diarrhea, nephritis, and encephalitis. Natural infection occurs via the oral route, and as a consequence, gut immunity plays an important role in restricting the dissemination of these pathogens. Studies from our laboratory have reported that the pathogens induce a rapid intraepithelial lymphocyte (IEL) response important for host protection. Although mucosal dendritic cells (DC) are likely involved in triggering an antigen-specific IEL response, the specific subset(s) responsible has yet to be identified. Toward this goal, we demonstrate a very important role for mucosal CD11b(-) CD8(+) DC in the initiation of an antigen-specific IEL in vivo. Effectively, after Encephalitozoon cuniculi infection, CD11b(-) CD8(+) DC were activated in the lamina propria (LP) and acquired the ability to process retinoic acid (RA). However, this subset did not produce interleukin 12 (IL-12) but upregulated CD103, which is essential for migration to the mesenteric lymph nodes (MLN). Interestingly, CD103(+) CD11b(-) CD8(+) DC in the MLN, in addition to processing RA, also secreted IL-12 and were responsible for gut imprinting specificity on mucosal CD8 T cells. To the best of our knowledge, this is the first report describing the importance of MLN CD103(+) CD11b(-) CD8(+) DC isolated from infected animals in the generation of an IEL response against a live pathogen.

Behavior and Properties of Mature Lytic Granules at the Immunological Synapse of Human Cytotoxic T Lymphocytes.

Killing of virally infected cells or tumor cells by cytotoxic T lymphocytes requires targeting of lytic granules to the junction between the CTL and its target. We used whole-cell patch clamp to measure the cell capacitance at fixed intracellular [Ca2+] to study fusion of lytic granules in human CTLs. Expression of a fluorescently labeled human granzyme B construct allowed identification of lytic granule fusion using total internal reflection fluorescence microscopy. In this way capacitance steps due to lytic granule fusion were identified. Our goal was to determine the size of fusing lytic granules and to describe their behavior at the plasma membrane. On average, 5.02 ± 3.09 (mean ± s.d.) lytic granules were released per CTL. The amplitude of lytic granule fusion events was ~ 3.3 fF consistent with a diameter of about 325 nm. Fusion latency was biphasic with time constants of 15.9 and 106 seconds. The dwell time of fusing lytic granules was exponentially distributed with a mean dwell time of 28.5 seconds. Fusion ended in spite of the continued presence of granules at the immune synapse. The mobility of fusing granules at the membrane was indistinguishable from that of lytic granules which failed to fuse. While dwelling at the plasma membrane lytic granules exhibit mobility consistent with docking interspersed with short periods of greater mobility. The failure of lytic granules to fuse when visible in TIRF at the membrane may indicate that a membrane-confined reaction is rate limiting.

Single-molecule microscopy of molecules tagged with GFP or RFP derivatives in mammalian cells using nanobody binders.

With the recent development of single-molecule localization-based superresolution microscopy, the imaging of cellular structures at a resolution below the diffraction-limit of light has become a widespread technique. While single fluorescent molecules can be resolved in the nanometer range, the delivery of these molecules to the authentic structure in the cell via traditional antibody-mediated techniques can add substantial error due to the size of the antibodies. Accurate and quantitative labeling of cellular molecules has thus become one of the bottlenecks in the race for highest resolution of target structures. Here we illustrate in detail how to use small, high affinity nanobody binders against GFP and RFP family proteins for highly generic labeling of fusion constructs with bright organic dyes. We provide detailed protocols and examples for their application in superresolution imaging and single particle tracking and demonstrate advantages over conventional labeling approaches.

Ubiquitous Autofragmentation of Fluorescent Proteins Creates Abundant Defective Ribosomal Products (DRiPs) for Immunosurveillance.

Green fluorescent protein (GFP) and other fluorescent proteins are essential tools for biological research. When fused to peptides or proteins as a reporter, GFP enables localization and quantitation of gene products in otherwise unmanipulated live cells or organisms. We previously reported that a sizable fraction of nascent GFP is post-translationally converted into a 20-kDa Triton X-100-insoluble proteasome substrate (Qian, S. B., Princiotta, M. F., Bennink, J. R., and Yewdell, J. W. (2006) J. Biol. Chem. 281, 392-400; Dolan, B. P., Li, L., Veltri, C. A., Ireland, C. M., Bennink, J. R., and Yewdell, J. W. (2011) J. Immunol. 186, 2065-2072). Here, we show that a similarly sized fragment is generated by all GFP and red fluorescent protein family members we examined. We demonstrate that fragmentation is a by-product of GFP chromophore rearrangement. A non-rearranging GFP mutant fails to fragment and generates diminished levels of K(b)-SIINFEKL complexes when SIINFEKL is genetically fused to either the C- or N-terminal domains of GFP fusion proteins. Instructively, another fragmenting GFP mutant that cannot create the functional chromophore but still generates fragments also demonstrates diminished K(b)-SIINFEKL generation. However, the mutant and wild-type fragments differ fundamentally in that wild-type fragments are rapidly liberated from the intact molecule and degraded quickly, accounting for increased K(b)-SIINFEKL generation. In the fragmenting mutant, the fragments are generated slowly and remain associated, likely in a native conformation based on their original structural description (Barondeau, D. P., Kassmann, C. J., Tainer, J. A., and Getzoff, E. D. (2006) J. Am. Chem. Soc. 128, 4685-4693). The wild-type GFP fragments represent the first biochemically defined natural defective ribosomal products to contribute peptides for immunosurveillance, enabling quantitation of peptide generation efficiency from this source of defective ribosomal products. More broadly, given the wide use of fluorescent proteins, their ubiquitous and abundant fragmentation must be considered when interpreting experiments using these extremely useful probes.

A robust pipeline for rapid production of versatile nanobody repertoires.

Nanobodies are single-domain antibodies derived from the variable regions of Camelidae atypical immunoglobulins. They show promise as high-affinity reagents for research, diagnostics and therapeutics owing to their high specificity, small size (∼15 kDa) and straightforward bacterial expression. However, identification of repertoires with sufficiently high affinity has proven time consuming and difficult, hampering nanobody implementation. Our approach generates large repertoires of readily expressible recombinant nanobodies with high affinities and specificities against a given antigen. We demonstrate the efficacy of this approach through the production of large repertoires of nanobodies against two antigens, GFP and mCherry, with Kd values into the subnanomolar range. After mapping diverse epitopes on GFP, we were also able to design ultrahigh-affinity dimeric nanobodies with Kd values as low as ∼30 pM. The approach presented here is well suited for the routine production of high-affinity capture reagents for various biomedical applications.

Fluorochrome-based definition of naturally occurring Foxp3(+) regulatory T cells of intra- and extrathymic origin.

Under physiological conditions, studies on the biology of naturally induced Foxp3(+) Treg cells of intra- and extrathymic origin have been hampered by the lack of unambiguous markers to discriminate the mature progeny of such developmental Treg-cell sublineages. Here, we report on experiments in double-transgenic mice, in which red fluorescent protein (RFP) is expressed in all Foxp3(+) Treg cells, whereas Foxp3-dependent GFP expression is exclusively confined to intrathymically induced Foxp3(+) Treg cells. This novel molecular genetic tool enabled us to faithfully track and characterize naturally induced Treg cells of intrathymic (RFP(+) GFP(+) ) and extrathymic (RFP(+) GFP(-) ) origin in otherwise unmanipulated mice. These experiments directly demonstrate that extrathymically induced Treg cells substantially contribute to the overall pool of mature Foxp3(+) Treg cells residing in peripheral lymphoid tissues of steady-state mice. Furthermore, we provide evidence that intra- and extrathymically induced Foxp3(+) Treg cells represent distinct phenotypic and functional sublineages.