We show that CD28 stimulation in the absence of TCR strongly up-regulates IL-22 gene expression and secretion. As recently observed for IL-17A, we also found that CD28-mediated regulation of IL-22 transcription requires the cooperative activities of both IL-6-activated STAT3 and RelA/NF-κB transcription factors. CD28-mediated IL-22 production also promotes the barrier functions of epithelial cells by inducing mucin and metalloproteases expression. Finally, by using specific inhibitory drugs, we also identified CD28-associated class 1A phosphatidylinositol 3-kinase (PI3K) as a pivotal mediator of CD28-mediated IL-22 expression and IL-22–dependent epithelial cell barrier functions.IL-22 is a member of the IL-10 cytokine family involved in host protection against extracellular pathogens, by promoting epithelial cell regeneration and barrier functions. Dysregulation of IL-22 production has also frequently been observed in acute respiratory distress syndrome (ARDS) and several chronic inflammatory and autoimmune diseases. We have previously described that human CD28, a crucial co-stimulatory receptor necessary for full T cell activation, is also able to act as a TCR independent signaling receptor and to induce the expression of IL-17A and inflammatory cytokines related to Th17 cells, which together with Th22 cells represent the main cellular source of IL-22. Here we characterized the role of CD28 autonomous signaling in regulating IL-22 expression in human CD4 We found that IL-22 gene expression and secretion was strongly up-regulated by CD28 in human CD4+ T cells were enriched from PBMC by negative selection using an EasySepTM isolation kit  and cultured in RPMI 1640 supplemented with 5% human serum , L-glutamine, penicillin and streptomycin. The purity of the sorted population was 95% to 99%, as evidenced by staining with anti-CD3 plus anti-CD4 Abs. Human naïve CD4+CD45RA+ and effector/memory CD4+CD45RO+ T cells were sorted using a high speed cell sorter . Purity of sorted cells was consistently > 98%, and was acquired on a cytometer . PBMCs were derived from buffy coats or anonymous healthy blood (HD) donors provided by the Policlinico Umberto I . Written informed consent was obtained from blood donors and both the informed consent form and procedure was approved by the Ethics Committee of Policlinico Umberto I . CACO-2 epithelial cell line from human colon was provided by ATCC  and cultured in DMEM supplemented with 10% FBS , L-glutamine, penicillin and streptomycin.Human primary CD4−1), anti-human CD3 , goat anti-mouse , anti-human CD3-PE (#555333), anti-human CD45RA BV421 (#562885), mouse anti-human STAT3 (#610189), anti-human CD45RO PE (#555493) (BD Biosciences); rabbit anti-human Lck , rabbit anti-human RNA polymerase II , anti-human RelB , anti-human GAPDH (#sc-25778), anti-human mucin 1 (MUC1) (#sc-7313) , rabbit anti-human RelA (#8242S) , mouse anti-human IL-6 , mouse anti-human IL-22  , anti-human CD4 FITC . Human recombinant IL-6 (rIL-6) was from Miltenyi Biotec . The following inhibitory drugs were used: PS1145 , S31-201 , AS605240 .The following antibodies were used: anti-human CD28  in 24 trans-well plates as indicated at 37°C. At the end of incubation, total cell extracts were obtained by lysing cells for 30 min on ice in 1% Nonidet P-40 lysis buffer , 1 mM EGTA, 1 mM MgCl2, 50 mM NaF, 10 mM Na4P2O7) in the presence of inhibitors of proteases and phosphatases . Proteins were resolved by SDS-PAGE and blotted onto nitrocellulose membranes. Blots were incubated with the anti-MUC1 or anti-GAPDH (1:400 dilution), extensively washed and after incubation with horseradish peroxidase (HRP)-labeled goat anti-rabbit  or HRP-labeled goat anti-mouse  developed with the enhanced chemiluminescence’s detection system . Protein levels were quantified by densitometric analysis using the ImageJ 1.50i program .CACO-2 cells were plated at 2.5 × 10+ T cells were plated at 2 × 106 ml−1 in 24-well plate or 24-well plate inserts in the experiments of co-culture with CACO-2 cells (2.5 × 105 ml−1) and stimulated for the indicated times with control isotype Abs (Ig) or crosslinked anti-CD28 (2 μg ml−1), or anti-CD3 (2 μg ml−1) or anti-CD3 plus anti-CD28 Abs (2 μg ml−1). Secretion of human IL-22, IL-6, and MMP9 was measured from the supernatants of CD4+ T cells cultured alone or with CACO-2 cells and stimulated with control isotype Abs or crosslinked anti-CD28.2 (2 μg ml−1), by using human IL-6 (#DY206), IL-22 (#DY782), and MMP9 (#DY911) ELISA kits (R&D Systems). Data were analyzed on a Bio-Plex . The assays were performed in duplicate. The sensitivity of the assay was 9.4 pg ml−1 for IL-6 and 31.2 pg ml−1 for IL-22 and MMP9.CD4+ T cells were plated at 2 × 106 ml−1 in 24-well plate or 24-well plate inserts in the experiments of co-culture with CACO-2 cells and stimulated for the indicated times with control isotype matched Abs (Ig) or crosslinked anti-CD28 (2 μg ml−1), or anti-CD3 (2 μg ml−1) or anti-CD3 plus anti-CD28 Abs (2 μg ml−1). Total RNA was extracted by either CD4+ T cells or CACO-2 cells using Trizol according to the manufacturer’s instructions and was reverse-transcribed into cDNA by using Moloney murine leukemia virus reverse transcriptase . TaqMan Universal PCR Master Mix and human IL-6, IL-22, MMP9, MUC1, and GAPDH primer/probe sets were from Thermo Fisher Scientific. The relative quantification was performed using the comparative CT method. The results were expressed as arbitrary units (AU) by using the mean value of cells stimulated with control Ig as CT calibrator or fold induction (F.I.) over control Ig-stimulated cells after normalization to GAPDH.CD4−644 of the human IL-22 promoter, the pIL-22 (-644)-GFP construct was obtained by subcloning the PCR fragment into AseI-Hind III sites within the CMV promoter of pEGFP-N1 vector . The sequence of human IL-22 promoter fragment (−644/+156) was verified by DNA sequencing and was as follow: 5′-ATTAATACAATTTTAAGATATATTTACTTCTGCCTTAATTGTTATGATCACTTAAAAATAGTTCCAAAAAGGGAAGAAAACAATAATTAGATTAGCCAAGACAGTTATTTTTGAAACATAAGTCTGGTTTAGAATTCAGCATGTTTAAAAATGAGATAAAATTATTTTAATAATGGAATGATCTGTTAGCTGTCATTACCATTTACTTTAAAGCAGAGGATATAGGACATGGGTCCTTTTTTTCTGATCACCTCCAATGAGATAAGAATCTATAAAGCTGGTAGGAAAATGAGTCCGTGACCAAAATGCTTACTCGGTCACTATAGGAGATCAAAACATTTTATACTAAATCTGAACTCTACTAAGACAAAACAATTGTGTTCTTTGAAAAATATGTAGGGTTTAGAAAATTTCTGGGATTTGTCTGTAAAATACCCTCCGGGCTCTAATAGTGACGTTTTAGGAAAACACTTGCATCTCAAGGTGGAAAGGATAGAGGTGGTGTTTTGTGGGCTCCTGTGGTGGTTAGGTCGTTCTCAGAAGACAGTACTGGAAATTAGATAATTGCTGATGTCATATTTTTCACAATTAAAAAAAAGTCAGTATCCTGGGGGCTATAAAAGCAGCAGCTTCTACCTTCCCCGTCACAAGCAGAATCTTCAGAACAGGTAGGCGTTTCGGCAAACTTGGTACAATTGGTTAGTTTGACGAAATACTTCTTGACTAATTTTGTTCCTTCACGTTGTCTTCGACCAGGTTCTCCTTCCCCAGTCACCAGTTGCTCAAGTTAGAATTGTCTGCA-3′.The pIL-22 (-644)-GFP construct containing the GFP under the control of the −644 bp region upstream of the transcriptional start site and the +156 region upstream the translational start site was generated as previously described . BrieflyPlasmid vectors expressing HA-tagged human RelA, IKKα, IKKβ and NIK were previously described , 38.3 GFP-positive events by gating for SSC and FSC.CD28-positive Jurkat T cells were electroporated  in 0.45 ml RPMI-1640 supplemented with 10% FBS with 1 μg of pIL-22(-644)-GFP together with pcDNA3 control vector 10 μg HA-RelA, or HA-IKKα or HA-IKKβ or 20 μg HA-NIK. Twenty-four hours after transfection, the cells were analyzed by using a BD FACScan flow cytometer . Mean fluorescence intensity was calculated on a total of 5 × 107 CD4+ T cells were stimulated as indicated and chromatin immunoprecipitation (ChIP) assays were performed as previously described  was performed for the human IL-22 promoter. Specific enrichment was calculated as previously described ((Ct of control ChIP-Ct of control Input)/2(Ct of specific ChIP - Ct of specific Input). The human IL-22 promoter primers used for each specific ChIP were as follow: RelA I, RelB I and pSTAT3 I, 5′-GCTTACTCAGCCACTATAGGAGATCA-3′ and 5′-CCGGAGGGTATTTTACAGACAAATCC-3′; RelA II, 5′-ACCCTCCGGGCTCTAATAGTGAC-3′ and 5′-AGAACGACCTAACCACCACAGGA-3′; pSTAT3 II and Pol II 5′-CCTGTGGTGGTTAGGTCGTTCT-3′ and 5′-GCTGCTTTTATAGCCCCCAGGAT-3′.10escribed . Brieflyescribed  by using−1). CACO-2 cells were plated at 2.5 × 105 cells ml−1 in 24-well plates and treated with 10 μM AS605240 or DMSO, as vehicle control, for 48 h. Cytotoxicity was analyzed by a BD Biosciences FACScalibur  by quantifying the percentage of PI positive cells. Results were calculated from at least three independent experiments.The cytotoxicity of AS605240 on CACO-2 cells was evaluated by propidium iodide (PI) staining  was performed to evaluate differences between continuous variables through Prism 5.0  using Student t test. For multiple group comparisons, significant differences were calculated using nonparametric Mann–Whitney + T cells from HD, relapsing-remitting MS (RRMS) and T1D patients (+ T cells from stable RRMS patients (+ T cells from HD with an agonistic anti-CD28 Ab (CD28.2) binding the same epitope recognized by B7 molecules (+ T cells from one representative HD induced a significant increase (p < 0.01) of IL-22 gene expression within 6 h that further increased 24 to 48 h and decreased 72 h after stimulation (+ T cells from a larger sample size (n = 7) (+ T cells from HD. As we previously observed for IL-17A (+CD45RA+ (+CD45RO+ (We have recently found that CD28 stimulation induces the expression of Th17 related cytokines in CD4patients –34. Morepatients . In ordeolecules  or anti- (n = 7) . Consist (n = 7) , CD28 str IL-17A  and other IL-17A , the ana+ T cells in a IL-6–dependent manner.These data evidence that CD28 intrinsic signaling regulates IL-22 gene expression and secretion in both naïve and effector/memory CD4+ T cells stimulated with agonistic anti-CD28.2 Abs. To do that, three distinct oligonucleotide probes were used; probe I (−338 to −203) for both STAT3 I and NF-κB I binding sites, probe II (−213 to −110) for NF-κB II binding site and probe III (−131 to −20) for both STAT3 II binding site and TATA box (+ T cells from a larger sample size (n = 9) and was associated to CD28-induced transcriptional activation of IL-22 promoter, as evidenced by RNA polymerase II (pol II) promoter occupancy (The human IL-22 gene promoter contains two putative STAT3 responsive elements (STAT3 I and II) and two NF-κB binding sites (NF-κB I and II) upstream of the transcription start site that have been involved in IL-22 promoter trans-activation , 43 Fi, 44 and,ccupancy , 46 and ccupancy  , or anti-CD28, or anti-CD3 or anti-CD3 plus anti-CD28 Abs and co-cultured in trans-well plates with CACO-2 cells for different times. The gene expressions of mucin 1 (MUC1), MMP9 and S100A9 anti-microbial peptide were then analyzed. The kinetic analysis evidenced that both MMP9 . Moreover, the up-regulation and MUC1 gene expression induced by co-culturing CACO-2 cells with CD28-stimulated CD4+ T cells was also associated with a strong increase of MUC1 protein content  required for epithelial barrier functions and protection against extracellular pathogens , 9. We nreported . CD4+ T + T cells   , 58 and  (RORγt) , 4. Here (RORγt) Figure 1 kinetic  and no s T cells , stimulapression , 60 resupression –63.+ T cells. We also found that activated STAT3 was also recruited to the proximal human IL-17A promoter and induced its trans-activation in response to CD28 stimulation . The patients/participants provided their written informed consent to participate in this study.MK performed most of the experiments, analyzed the data, interpreted the results, and helped in writing the manuscript. CA, SF, SC, MS, and MB performed parts of the experiments and data analyses. SA contributed with human samples for the study. MK, CA, SF, MS, and LB were involved in the discussion about the data. LT designed the study, coordinated the work, and wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by: the Italian Foundation for Multiple Sclerosis (FISM 2016/R/29), “Progetto Ateneo”  and Istituto Pasteur Italia-Fondazione Cenci Bolognetti  to LT; the Italian Ministry of Health  and the Italian Foundation for Multiple Sclerosis  to LB; the Italian Ministry of Health (GR-2016-02363725 and GR-2018- 12365529) to MS.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.


Fluorogen-activating proteins (FAPs) are innovative fluorescent probes combining advantages of genetically-encoded proteins such as green fluorescent protein and externally added fluorogens that allow for highly tunable and on demand fluorescent signaling. Previously, a panel of green- and red-emitting FAPs has been created from bacterial lipocalin Blc (named DiBs). Here we present a rational design as well as functional and structural characterization of the first self-assembling FAP split system, DiB-splits. This new system decreases the size of the FAP label to ~8–12 kDa while preserving DiBs’ unique properties: strong increase in fluorescence intensity of the chromophore upon binding, binding affinities to the chromophore in nanomolar to low micromolar range, and high photostability of the protein-ligand complex. These properties allow for use of DiB-splits for wide-field, confocal, and super-resolution fluorescence microscopy. DiB-splits also represent an attractive starting point for further design of a protein-protein interaction detection system as well as novel FAP-based sensors. The central idea is that reconstitution is followed by regain of a specific function which is abolished in the separated parts. Theoretically, many proteins can be divided into such fragments. In practice, the identification of a functional split protein is still nontrivial, although some success in direct evolution-basedin vivo protein-protein interaction detection4. Successful cleavage of the reporter protein, dihydrofolate reductase, fused to the C-terminal fragment of ubiquitin was happening only when both the C-terminal and mutated N-terminal fragments of ubiquitin were expressed as fusions to a leucine zipper homodimerization domain but not when expressed individually.Split proteins were first employed when using ubiquitin for 5, ß-lactamase6, thymidine kinase7, or luciferase8. This allows for real time and quantitative analysis of protein interactions in vitro as well as in model organisms. The desire for more user-friendly methods for detecting protein-protein interactions in complex environments and for identification of their precise cellular localization in combination with enormous progress in fluorescent microscopy techniques prompted the creation of fluorescent split proteins. This included split versions of green fluorescent protein (GFP)10, its differently colored derivatives and homologs13, far-red emitting phytochrome-based fluorescent proteins14, or even dual split reporters15.Later, this concept was applied to a number of other proteins. Many of them were enzymes like dihydrofolate reductase12.When used for protein-protein interaction detection, spontaneous self-association of split proteins is highly undesirable. Such self-association events will contribute to the false positive signal and decrease the overall sensitivity of the method. However, spontaneously self-complementing fluorescent split pairs were found to be useful. Their usage allows for substantial decrease of the tag size that is required to be fused to the protein of interest. Therefore, it diminishes potential influence of the tag on the protein of interest behavior16, IFP1.417, iRFP18, and UnaG19 find their ligands  readily available in mammalian cells. Other FAPs like various dye-binding antibodies21, FAST22, DiBs23, and de novo computationally designed mFAPs24 require an exogenous supply of the chromophore. The latter group of FAPs provides multiple benefits. First, available synthetic molecules show a wide range of chemical and photophysical properties allowing for the creation of fluorescent probes with a desired combination of characteristics. Second, external addition of the ligand gives control over the timing and intensity of the fluorescent signal. Third, the noncovalent nature of interaction provides, in some systems, millisecond-scale blinking of the fluorescent signal caused by ligand binding-dissociation events. In these cases, the optimal signal density for high-resolution image reconstruction can be achieved simply by using an appropriate dye concentration. This approach circumvents usage of damaging levels of illumination intensities, which is common for many single-molecule localization microscopy techniques25.Fluorogen-activating proteins (FAPs) are a group of unrelated proteins capable of binding to non-protein ligands (fluorogens) and increase the fluorescence quantum yield and/or change spectral properties of these ligands. Some of these FAPs like miniSOG14 and reversible26 split systems, photoactive yellow protein-based splitFAST27, a label for correlative light and electron microscopy split-miniSOG28, and a bilirubin-binding UnaG-based split reporter uPPI29. Two of these FAP-based splits, IFP PCA26 and splitFAST27, require exogenous supply of the chromophore for imaging in eukaryotic cells. It also has been shown that the full-length UnaG fluorescence recovery under photobleaching conditions needs excess bilirubin added into the solution30. While IFP chromophore, biliverdin, forms a covalent adduct with the protein, only splitFast and uPPI seem to be suitable for use with binding and dissociation events-detecting single-molecule localization microscopy techniques like protein-PAINT23. However, to the best of our knowledge, such an application has not been shown yet for either probe. The reported usage of split-miniSOG for imaging at subdiffraction resolution via electron microscopy is limited to fixed samples.Following the extension of the list of reported FAP systems, FAPs-based splits, also known as bimolecular fluorescence complementation (BiFC) systems, begin to appear. That includes multiple bacteriophytochromes-based irreversiblein vitro screenings we created a panel of FAPs from bacterial lipocalin Blc (named DiBs) capable of recovering the fluorescence of synthetic analogs of green and red fluorescent proteins’ chromophores31. Here we report on DiB-splits, a self-assembling FAP split system which has been inspired by the domain-swapped structure of a full-length DiB protein. This new FAP system reduces the size of the label needed to be conjugated with a protein of interest to ~8–12 kDa and is compatible with wide-field, confocal, and super-resolution fluorescence microscopy. DiB-splits also offer an attractive template for design of a protein-protein interaction detection split system as well as FAP-based sensors.In our previous work, using a combination of computational and 32, previously characterized wild type Blc (wtBlc) protein35, as well as another Blc mutant, DiB1, that has been co-crystallized with the M739 . Our attempts to structurally characterize other DiB proteins23 in apo and bound states resulted in obtaining protein crystals of DiB3 in the apo form at low pH conditions (pH 3.5) which diffracted to 1.6 Å. The asymmetric unit contains only one protein chain. However, it forms a biological assembly (dimer) with a crystallographic symmetry mate. The intertwined dimer is caused by domain swapping: each of the two Blc-like eight-stranded beta-barrel folds is created by the N-terminus of one of two polypeptide chains and the C-terminus of the other , except for the region that connects the exchanging parts of the protein .The lipocalin fold contains a single eight-stranded continuously hydrogen-bonded antiparallel β-barrel complemented by an α-helix. This common fold has been observed for other lipocalin protein family membersher Fig. . As obsein cellulo protein labelling23, we proposed that the observed domain swapping was driven mainly by the very low pH conditions of the crystallization buffer rather than the private DiB3 mutations (V74F and L141Q). To further evaluate this assumption, we calculated the interaction energies between N- and C-termini fragments of the wtBlc, DiB1, and DiB3 proteins using Rosetta38. Despite the fact that wtBlc protein seems to be slightly more stable, DiB3 is not an outlier . Such property is crucial for the successful creation of a split system. Usually designing a split protein involves laborious screening of multiple protein sites in order to select an appropriate cutting point First, we tested whether N- and C-termini fragments, created by separation of a protein chain in the hinge region, are indeed capable to form the lipocalin-like structure when brought together. We fused each of two fragments of DiBs1–3 with one of two leucine zipper peptides as shown on Fig. 35 which locks side chains in the preferable conformation for ligand binding. The opposite effect, weaker binding seen in the case of the red-shifted DiB3-split-Zip:M739 complex, supports the previously suggested hypothesis of the alternative binding mode of the ligand in that complex .Split-Zip proteins showed properties similar to the “parental” full-length variants upon addition of the M739 fluorogen including binding affinities, fluorescence spectra maxima, and extinction coefficients Table  and S5. 43. On the other hand, in the case of spontaneous self-assembly, the split form of the labelling system allows for a reduction in the size of the labelling tag that needs to be added to a protein of interest. Hence, it minimizes tags’ influence on that protein44. Therefore, as a next step we checked the ability of the DiB N- and C-termini fragments to self-assemble. For this we deleted the leucine zippers as shown on Fig. The behavior of a split system in the absence of additional “attracting force” like leucine zippers or other interacting proteins determines the range of its possible applications. If the assembly of the split protein is conditional (fails to self-assemble spontaneously), it can be used for investigation of protein-protein interactionsSelf-assembling split proteins retain their ability to bind and increase fluorescence of the fluorogen M739 Table  and S5, 35 as well as a functional dimer34. However, according to our knowledge, the possibility of a trimer formation was never investigated before. According to size-exclusion chromatography conducted during purification routine of split proteins there were no signs of oligomerization of any kind . The residues of the hinge loop and two adjacent β-strands are also responsible for the main difference between wtBlc and DiB2-split monomers . We assessed the behavior of these constructs in separate transfections. Cells transfected with the TagBFP-splitC110–177 construct alone showed uniform distribution of the blue fluorescence signal throughout cytoplasm and nuclei of the cells  produced uniformly distributed blue fluorescent signal  TagBFP-splitN1–125 + TagBFP-splitC126–177 and (2) TagBFP-splitN1–125 + TagBFP-splitC110–177. Upon addition of the M739 chromophore, recovery of the DiB2-specific fluorescent signal in green channel was observed only in the second pair . We speculate that this can be caused by somehow compromised integrity of the new splitN1–125 fragment. For example, because of presence of an alternative conformation of the new C-terminus. Combination of two protein fragments with partial sequence overlap seems to allow for more efficient assembly or/and longer half-time of the functional complex.Next, we tested the ability of the split system to function 1–125 and splitC110–177 fragments in fusion with either histone H2B or vimentin, and conventional fluorescent protein TagBFP. The assembled split would be visible in a distinct localization in green detection channel if self-assembly occurs successfully. The blue detection channel would show the overall distribution of one of the split halves within a cell and allow for detection of aggregation, non-assembled portion, or other undesirable behavior. We first tested two pairs of H2B-fused proteins: (1) splitN1–125 fragment fused with H2B and splitC110–177 fused with TagBFP (H2B-splitN1–125 + TagBFP-splitC110–177), and (2) splitN1–125 fragment fused with TagBFP and splitC110–177 fused with H2B (H2B-splitC110–177 + TagBFP-splitN1–125). Upon transient transfection of the first pair of fusion proteins (H2B-splitN1–125 + TagBFP-splitC110–177), the blue fluorescent signal of the TagBFP confirmed absence of aggregation both in the nucleus and in the cytoplasm , DiB2-split can be used as a protein-PAINT tag: bursts of fluorescence from individual protein-ligand interactions are clearly detectable Fig.  of labelin vivo and was found to give bright and specific fluorescent signal indistinguishable from the one of the full-length protein.In this study, inspired by the obtained domain-swapped crystal structure of the DiB3 protein in its apo state, we designed and characterized a novel DiB-split FAP system. The two fragments of the split proteins were able to spontaneously reassemble fully restoring fluorogen-activating and spectral properties of the “parental” full-length DiBs. Crystallization of one of these proteins, DiB2-split, further corroborate the preservation of the lipocalin fold by the system. The DiB2-split was tested 23 label of a smaller size for super-resolution imaging in living cells. The decrease of the tag size provided through the split can diminish influence of the tag on the protein of interest. In vitro data suggest near complete assembly of DiB2 from split fragments, making DiB-splits a feasible replacement for full-length DiBs or fluorescent proteins in cases where the size of the molecular tag matters. Moreover, while DiB-split fluorescence does not require post-assembly chromophore maturation unlike self-assembling fluorescent proteins12, it can be used for immediate detection of different biological processes such as protein expression and early trafficking events or as a faster reporter of protein solubility.This DiB-split system presents a proof-of-principle demonstration of the potential of the lipocalin scaffold to create a split system. It is immediately applicable as a protein-PAINT46. Both systems do not require oxygen for their function and might be used in oxygen-deficient systems as it was previously shown with FAST47.DiB-splits as well as the parental full-length DiBs have lower signal-to-noise ratio in comparison with FAST family probes. However, FAST localization density decreased rapidly during data acquisition time in single-molecule localization microscopy regime and successful super-resolution imaging using FAST required protocol modifications and usage of oxygen scavengers1–109) as well as the elongated one (splitN1–125) can be redesigned for better stability. Moreover, mutagenesis of the new N- and C-termini could increase binding affinities for the fluorogen. In addition, there is potential for the design of a variety of other fluorescent tools. For example, redesign of the intramolecular interface of the DiB-split proteins reported here to promote higher independence of the N- and C-termini fragments can result in a new FAP-based tool for protein-protein interactions detection. Such tool would complement the existing mEos3.248 and PAmCherry1-based49 super-resolution imaging compatible BiFC labels providing additional benefit of fast and oxygen level independent measurements. Spatial proximity of the natural N- and C-termini of the protein makes DiB proteins a promising starting point for the design of DiB-based circularly permuted proteins. While the discovered split point is close to the ligand binding site, such circularly permuted proteins represent a promising starting point for DiB-based biosensors design. Successful permutation might also allow for the creation of a new self-assembling split system with smaller parts analogous to self-complementing split fluorescent GFP1110 and sfCherry1112 tags.DiB-splits would benefit from further optimization of the location of the split point. The original N-terminus fragment of the system  and C-fragments (residues 110 to 177) of the DiB mutants23 as well as leucine zipper peptides with adjacent upstream or downstream portions of the vector from the pMRBad-Z-CspGFP and pET11a-Z-NspGFP plasmids, correspondingly, and the upstream portion of the pET11a-Z-NspGFP plasmid including His-tag. Second, we used the overlap PCR to create DNA fragments containing leucine zipper peptides fused with N- or C-fragments of the DiB mutants flanked by upstream and downstream portions of the vector. These fragments were digested with BamHI and XbaI or NcoI and BsrGI restriction enzymes and ligated in the original vectors. These vectors were further used to create split fragments without leucine zipper peptides. For this we amplified N-fragments of the DiB mutants with adjacent upstream portion of the vector introducing stop codon and BamHI restriction site instead of leucine zipper peptide coding sequence and C-fragments of the DiB mutants introducing start codon and NcoI restriction site instead of leucine zipper peptide. The PCR products were again digested with BamHI and XbaI or NcoI and BsrGI restriction enzymes and ligated in the original vectors.Plasmids pMRBad-Z-CspGFP (Addgene plasmid #40730) and pET11a-Z-NspGFP (Addgene plasmid #40729)53. The resulted constructs’ amino acid sequences are provided below. The linker sequences are underlined.DiB2-split fusions with H2B, vimentin, and TagBFP were generated by Golden Gate Assembly1–125>H2B-splitNDPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRFERGLEKVTATYSLRDDGGLNVINKGYNPDRGMWQQSEGKAYFTGAPTRAALKVSFFGPFYGGYNVIALDREYSG*MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVLKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPGELAKHAVSEGTKAITKYTSAK110–177>TagBFP-splitCDPPVATMGPFYGGYNVIALDREYRHALVCGPDRDYLWILSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGSG*MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN110–177>H2B-splitCDPPVATMGPFYGGYNVIALDREYRHALVCGPDRDYLWILSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGSG*MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVLKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPGELAKHAVSEGTKAITKYTSAK1–125>TagBFP-splitNDPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRFERGLEKVTATYSLRDDGGLNVINKGYNPDRGMWQQSEGKAYFTGAPTRAALKVSFFGPFYGGYNVIALDREYSG*MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN1–125>vimentin-splitNDPPVATGMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRFERGLEKVTATYSLRDDGGLNVINKGYNPDRGMWQQSEGKAYFTGAPTRAALKVSFFGPFYGGYNVIALDREYSG*MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEENFAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLEG110–177-TagBFP>splitCDPPVATMSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN*MGPFYGGYNVIALDREYRHALVCGPDRDYLWILSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGSG1–109>TagBFP-splitNDPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRFERGLEKVTATYSLRDDGGLNVINKGYNPDRGMWQQSEGKAYFTGAPTRAALKVSFFSG*MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN126–177>TagBFP-splitCDPPVATMRHALVCGPDRDYLWILSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGSG*MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN>vimentin-DiB2DPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRFERGLEKVTATYSLRDDGGLNVINKGYNPDRGMWQQSEGKAYFTGAPTRAALKVSFFGPFYGGYNVIALDREYRHALVCGPDRDYLWILSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGSG*MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEENFAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLEGCorrectness of all obtained constructs was confirmed by sequencing.E. coli strain. Cells were grown in LB media supplemented with 100 µg/mL ampicillin (full-length DiB proteins) or 100 µg/mL ampicillin and 50 µg/mL kanamycin (split-Zip and split proteins) at 37 °C. Expression was induced by addition 0.04% L-arabinose (full-length DiB proteins) or 0.2% L-arabinose and 10 μM IPTG (split-Zip and split proteins) at 0.8 OD. Cells were harvested after 3 hours of expression at 37 °C and were resuspended in PBS buffer, pH 7.4. Suspensions were frozen at −80 °C and thawed at room temperature three times. DNA was destroyed by short sonication and the lysates were centrifuged to obtain cell-free extracts. The proteins were first purified using gravity flow columns with TALON metal affinity resin (Clontech) and further purified by size-exclusion chromatography on a HiLoad 16/600 Superdex 75 pg or Superdex 200 pg 10/300 GL column  pre-equilibrated with 50 mM sodium phosphate buffer, pH 6.0.All proteins were expressed in XJb(DE3) Autolysis (Zymo Research) 54 colorimetric assay (Bio-Rad) and bovine serum albumin standard. Single point absorption measurements (595 nm) were performed using FlexStation 3 microplate reader (Molecular Devices). All measurements were performed in triplicate.Protein concentrations were estimated using the Bradford dye-binding method-basedHoriba Jobin Yvon Fluoromax-3 fluorometer was used to detect full fluorescence excitation and fluorescence emission spectra for excitation/emission maxima evaluation.Fluorescence quantum yield was measured relative to a known standard keeping all instrumental conditions identical. Previously characterized DiB:M739 complexes as well as free M739 chromophore were used as standards. Absorbance spectra were detected using double-beam Shimadzu UV-1800 UV/Vis spectrophotometer. Fluorescence spectra were measured using Horiba Jobin Yvon Fluoromax-3 fluorometer.23 using FlexStation 3 microplate reader (Molecular Devices). In brief, constant amount of the chromophore solution was added to protein solutions of different concentrations. The full fluorescence emission spectra were collected using wavelength close to protein-chromophore complex excitation spectrum maximum wavelength. Fluorescence intensity at complex emission spectrum maximum wavelength was extracted and used to determine apparent dissociation constants (Kd).Titrations were performed and analyzed as previously describedDiB3  was crystalized at 21 °C in 0.8 M sodium citrate, 50 mM sodium borate, 0.1 sodium acetate, pH 3.5 with protein to precipitant volume ratio of 1:1 using hanging drop vapor diffusion technique. Crystals grew within 1–3 days and were flash frozen in liquid nitrogen using Parabar 10312 oil as cryoprotectant.DiB2-split  crystals were obtained at 21 °C in 1.6 M ammonium sulphate, 0.1 M MES, pH 4.5 with protein to precipitant volume ratio of 1:1 or in 1.6 M ammonium sulphate, 0.1 M MES, pH 4.5 supplemented with 0.1 M Iron(III) chloride hexahydrate or 5% w/v n-Dodecyl-b-D-maltoside according to Hampton Research Additive Screen protocol using sitting drop vapor diffusion technique. Crystals grew within 1 week and were flash frozen in liquid nitrogen using Parabar 10312 oil as cryoprotectant.55. The crystal structures were solved by molecular replacement with MOLREP56 using the wtBlc structure (PDB ID 1QWD) as a search model. Models building and iterative refinement were performed with Coot57 and REFMAC58, respectively. The final statistics of the structures are shown in Supplementary Table S1. The models have been deposited into the Protein Data Bank (PDB ID 6UKK and 6UKL). Structure figures were prepared using PyMol .Diffraction data were collected at the Life Sciences Collaborative Access Team beamline 21-ID-G at the Advanced Photon Source, Argonne National Laboratory. The diffraction data were processed using xia2 software suite2. For transient transfections FuGENE 6 reagent (Roche) was used. Immediately before imaging DMEM was replaced with HHBS media (Hanks Buffer supplemented with 20 mM Hepes).HEK293 and HeLa Kyoto cells were grown in Dulbecco’s modification of Eagle’s medium (DMEM) (PanEco) supplied with 50 U/ml penicillin and 50 µg/ml streptomycin (PanEco), 2 mM L-glutamine (PanEco) and 10% fetal bovine serum  at 37 °C and 5% CO−2 of 488 nm laser light intensity. Typical acquisitions were 10 000 frames taken at a frequency of 30 Hz.Widefield fluorescence microscopy was performed with the Leica DMI6000B inverted microscope, Zyla 5.5 sCMOS camera (Andor), CoolLED pE-300 light source, GFP and BFP filter sets. Single-molecule localization super-resolution imaging of living cells was performed with Nanoimager S (ONI). Imaging in HILO mode was performed with  1.1 kW cm59.Localizations during acquisition were detected using NimOS 1.6.1.9898 . Super-resolution image reconstruction was performed using default values of photon, precision and sigma filters in NimOS. Data analysis was performed using a custom-made Python script. Image resolution was determined by decorrelation analysis pluginTo prepare DiB3 structure for analysis we reconstructed the intertwined dimer using crystallographic symmetry and saved the N-terminus portion of one protein chain (residues 24 to 109) and the C-terminus portion of the other protein chain (residues 114 to 175) as a single pdb file. Both wtBlc (PDB ID 1QWD) and DiB1 crystal structures contain two protein chains in the asymmetric unit. We separated the chains into different pdb files and used both for analysis. Each “original” chain was further separated into two chains mimicking DiB3-split and only residues 24 to 109, and 114 to 175 were left to make the length of all structures equal. Each of the 5 prepared structures was then relaxed and the interface energy between two chains was calculated using the following protocol:<ROSETTASCRIPTS > <SCOREFXNS/ > <TASKOPERATIONS/ > <MOVERS > <FastRelax name = “fastrelax” relaxscript = “default”/><ClearConstraintsMover name = “clear”/><InterfaceAnalyzerMover ligandchain = “B” name = “iface_analyzer” pack_input = “0” pack_separated = “1” packstat = “0” scorefxn = “REF2015” tracer = “0”/></MOVERS > <FILTERS/ > <APPLY_TO_POSE/ > <PROTOCOLS > <Add mover = “fastrelax”/><Add mover = “clear”/><Add mover = “iface_analyzer”/></PROTOCOLS > <OUTPUT scorefxn = “REF2015”/ > </ROSETTASCRIPTS > The protocol was repeated independently 100 times for each of the starting structures. The data obtained for two separate chains in the asymmetric unit from wtBlc and DiB1 crystal structures were combined. The distribution of the obtained interface energies between N- and C-terminus fragments is shown on Supplementary Fig. S2.Supplementary information.Video1Video2


N-glycans and high-mannose type N-glycans, suggests that high-mannose-specific seaweed lectins are particularly well adapted as glycan probes for coronaviruses. This review presents a detailed study of the carbohydrate-binding specificity of high-mannose-specific seaweed lectins, demonstrating their potential to be used as specific glycan probes for coronaviruses, as well as the biomedical interest for both the detection and immobilization of SARS-CoV-2 to avoid shedding of the virus into the environment. The use of these seaweed lectins as replication blockers for SARS-CoV-2 is also discussed.Seaweed lectins, especially high-mannose-specific lectins from red algae, have been identified as potential antiviral agents that are capable of blocking the replication of various enveloped viruses like influenza virus, herpes virus, and HIV-1 in vitro. Their antiviral activity depends on the recognition of glycoprotein receptors on the surface of sensitive host cells—in particular, hemagglutinin for influenza virus or gp120 for HIV-1, which in turn triggers fusion events, allowing the entry of the viral genome into the cells and its subsequent replication. The diversity of glycans present on the S-glycoproteins forming the spikes covering the SARS-CoV-2 envelope, essentially complex type  The occurrence of lectins, formerly designated as hemagglutinins due to their capacity to agglutinate red blood cells from humans and various animals, in marine seaweeds has been recognized for a long time, following the pioneering works of Boyd et al.  and BlunGriffithsia sp., exhibited inhibiting properties towards HIV-1 [Kappaphycus alvarezii (KAA-2) [Eucheuma serra (ESA-2) [Halimeda renschii (HRL-40) [N-glycans (HM-glycans) occurring at the surface of the influenza virus [Boodlea coacta (BCA) [Oscillatoria agardhii [Nostoc ellipsosporum [Microcystis aeruginosa [Scytonema varium [N-glycans associated to different glycoprotein receptors, e.g., hemagglutinin from influenza virus [Griffithsin (GRFT) from the red alga ds HIV-1 . High-ma (KAA-2) ,13, Euch (ESA-2) , and Hal(HRL-40)  also recza virus ,11,12,14za virus ,11,13. Tagardhii , cyanovisosporum ,19,20,21ruginosa ,23, and a varium . Antivira varium , influena varium ,19, hepaN-glycans decorating the spikes arrayed on the surface of the SARS-CoV-2 envelope, essentially comprised of complex N-glycans and high-mannose N-glycans [Taking into account the antiviral properties and the well known diversity of -glycans ,26, one SARS-CoV-2 spikes consist of homotrimers of S-glycoproteins and play a key role in both the recognition and the subsequent membrane fusion events, resulting in the infection of the host cells ,28. SpikN-glycans of the S-glycoproteins forming the SARS-CoV-2 spikes [Due to the high number of high-mannose 2 spikes ,26,35, hAccording to their carbohydrate-binding specificity towards simple sugars, (seaweed) lectins can been classified in five main groups of Man-specific lectins, GlcNAc-specific lectins, Gal/GalNAc-specific lectins, Fuc-specific lectins, and Sia-specific lectins. Until now, the research on seaweed lectins has focused especially on the Man-specific lectins, and these lectins have been characterized in more detail.To date, a large number of seaweed lectins have been screened, but only a few lectins have been studied in detail or have been characterized at the molecular/structural level. In spite of these limitations, the amino acid sequences and some structural information have become available for some Man-specific seaweed lectins from the red algae (Rhodophyta), the yellow-green algae (Ochrophyta), and the green algae (Chlorophyta) .-Galanthus nivalis agglutinin)-related family of lectins consists of protomers organized in a β-prism II or β-trefoil. The red alga Grateloupia chiangii lectin (GCL) and the green alga Boodlea coacta lectin (BCA) present this type of structural organization.The GNA , Nannochloropsis , Ostreococcus , and Porphyra .The legume lectin-related family is made of protomers organized in a β-sandwich or jelly roll fold (two β-sheets). This structural scaffold occurs in a few lectins from the genera Man-specific seaweed lectins belong to a few well defined protein families which have been previously identified and characterized for the molecular organization of their protomers, especially in plants :-The GNAN-acetyllactosaminic type glycans and high-mannose type glycans.Man-specific seaweed lectins readily accommodate Man, oligomannosides, and high-mannose type glycan chains. In addition, most of them recognize the tri-mannosyl core Manα1,3-Manα1,6-Man occurring in both Grateloupia chiangii lectin (GCL) offers a nice example of a Man-specific red alga lectin with a β-prism II structure. The β-prism II scaffold consists of three bundles of four antiparallel β-strands arranged into a flattened trefoil-shaped structure around a central pseudoaxis. The GCL lectin dimer consists of two covalently linked swapped protomers organized in a β-trefoil in such a way that both protomers become oriented almost orthogonally  with a β-prism II structure [The ogonally A. Each pogonally B,C. The tructure .Porphyra umbilicalis lectin (PUL) illustrates the β-sandwich organization of the lectin protomer, resulting from the covalent superposition of two strands of β-sheets connected by more or less extended loops, forming the front and back faces of the β-sandwich, respectively (Nannochloropsis gaditana (BU14), and in the green alga Ostreococcus tauri (OtL) [The ectively A,B. Thisectively B, form tectively C. The CBectively D. Two otri (OtL)  , and two additional stacking interactions with aromatic residues Y28 and Y110 C. The CBThe CBS of griffithsin also accommodates dimannosides, e.g., Manα1,6Man, via a similar network of 8 hydrogen bonds and stacking interactions with the Y28 and Y110 residues, but the second Man unit located at the reducing end of the disaccharide does not participate in the interaction A,B. A fr8) to the lectin  towards high-mannose glycans, has been studied in detail by Sato et al. [The carbohydrate-binding specificity of another Man-specific lectin with a β-prism I structure from the red alga o et al. . The hig5) to the modeled KAA-2 lectin, showed the existence of four CBS located at both ends of the β-barrel forming each protomer of the KAA-2 dimer , e.g., cyanovirin-N (CV-N) from the cyanobacterium sosporum , microviruginosa ,23, scyta varium , and theinin OAA . All of inin OAA .N-glycans of the N-acetyllactosaminic type  and three O-glycosylation sites T323, S325 and T678 are actually glycosylated [The spike S-glycoprotein consists of a single polypeptide chain of 1273 amino acids (140 kDa), containing 22 potential osylated ,35.N-glycosylation sites NXT/NXS are highlighted in dark blue and the O-glycosylation sites are shown in bold letters highlighted in yellow:As shown below the complete amino acid sequence of the S glycoprotein of SARS-CoV-2 is made of two S1 and S2 subunits. The RBD of subunit S1 is highlighted in green and the S1/S2 cleavage site for cathepsin and serine protease TMPRSS2 is highlighted in red. All the VNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGHHHHHHHHSAWSHPQFEKGGGSGGGGSGGSAWSHPQFEKN- and O-glycans attached to the potential N- and O-glycosylation sites decorating the amino acid sequence of the S-glycoprotein of SARS-CoV-2 , are almost exclusively occupied by often sialylated, bi-, tri- and tetra-antennary N-glycans of the complex type , and GalNAcGalNeuAc  A large diversity was observed in the types of N-glycans and high-mannose N-glycans. Moreover, high-mannose glycans predominantly occur at the top of the S-glycoprotein whereas complex N-glycans are localized at the bottom of the glycoprotein, close to the viral envelope surface.Bi- and tri-antennary glycans are predominantly represented, across all categories of the complex N-glycosylation sites, 331NIT and 343NAT, predominantly occupied by high-mannose N-glycans that should be readily accessible to Man-specific seaweed and cyanobacterial lectins.An interesting note is that the RBD , only contains two O-glycosylation sites T323 and S325, are rather buried at the top of the S-glycoprotein, in such a way that the O-glycans are not identified as key targets for the binding of GalNAc/T-Tn-specific lectins to the S-glycoprotein trimer. However, O-glycosylation site T678 is pretty well exposed at the bottom of the S-glycoprotein and therefore, should be accessible to the GalNAc/T-Tn-specific lectins. Accordingly, seaweed lectins with the corresponding specificity should not be relevant glycan probes for SARS-CoV-2, except for the single exposed O-glycosylation site T678  and two rather buried sites for O-glycosylation (T323 and S325)  . In addist cells ,35.The spikes S-glycoprotein trimers covering the SARS-CoV-2 virions, mediate the binding to the ACE2 receptor through their RBD (S1 subunit), and the subsequent fusion of the viral membrane with the cell membrane (S2 subunit). The spike S-glycoprotein exhibits some flexibility and conformational motions of the S-glycoprotein are pH-dependent . HoweverBoodlea coacta, were shown to possess antiviral properties against various enveloped virus including influenza, herpes, and hepatitis C viruses, and HIV-1  or gp120 (HIV-1) glycoproteins (n = 0) to 100% (n = 3) (A detailed study of the binding-activity towards pyridylaminated (PA-)-oligosaccharides measured for Man-specific lectins of red algae , green a coacta ) and theagardhii ), showedproteins . The bin (n = 3) .An interesting note is that some of the high-mannose glycans of both the hemagglutinin of the influenza virus and the gp120 of HIV-1 recognized by the lectins, also decorate the S-glycoprotein forming the spikes occurring at the surface of the SARS-CoV-2 envelope. Accordingly, the algal Man-specific lectins should similarly interact with the SARS-CoV-2 through the recognition of their spike S-glycoproteins. In this respect, griffithsin (GRFT) was shown to inhibit both the replication and cytopathy of the coronavirus SARS-CoV .Kappaphycus alvarezii), HRL-40 , BCA (Boodlea coacta) and OAA (Oscillatoria agadhii), shows they are the best exposed at the surface of the S-glycoprotein monomer ), N122, N234, N331, N343, N616 and N717 occupied by the high-mannose glycans recognized by the Man-specific lectins KAA-2  and seaweed lectins specific for terminal Neu5Ac residues, could interact with the S-glycoprotein of SARS-CoV-2  RS-CoV-2 . Looking at T678   [http://www.rcsb.org/pdb/) [Burkholderia oklahomensis (PDB code 4GU8 and 4GK9) [Oscillatoria agardhii (PDB code 3OBL) [The atomic coordinates of griffithsin GRFT from de 2GTY) , and lecde 2GTY) , 6α-mannde 2GTY)  and highde 2GTY) , were targ/pdb/) . Similarnd 4GK9)  and the de 3OBL)  were alsGrateloupia chiangii [Kappaphycus alvarezii [Porphyra umbilicalis [Homology modeling of other lectins including the β-prism II folded GCL from chiangii , the β-blvarezii , and theilicalis , was perilicalis  using vailicalis , ANOLEA ilicalis , and theilicalis ,97, wereDocking of simple sugars and oligosaccharides was performed with YASARA and SwissDock . HydrophGriffithsia sp., readily recognized the high mannose N-glycans located on the very similarly glycosylated SARS-CoV S-glyco- protein [The S-glycoprotein on the surface of the SARS-CoV-2 virus is a highly glycosylated protein. Due to the exposed localization of high-mannose glycans at the top of the S-glycoprotein trimers many of these glycans are readily accessible to carbohydrate-binding proteins. Seaweed lectins represent well adapted glycan probes for the specific recognition of this type of viruses. In this respect, the Man-specific lectin griffithsin (GRFT) of the red alga  protein ,63. More protein , GRFT waMoreover, the binding of seaweed lectins to SARS-CoV-2 virus could be applied in biomedical research, e.g., using Man-specific seaweed lectins (1) for detection purposes of the virus on various contaminated surfaces such as doorknobs or furniture elements, (2) as an efficient barrier to avoid the shedding into the environment of contaminating virions and, (3) as control reagents for the occurrence of viral particles in biotic/abiotic samples. Depending on the case, whether properly labelled, e.g., fluorochrome-labelled, Man-specific seaweed lectins could be used directly as glycan probes or unlabelled lectins could be further detected using properly labelled, e.g., fluorochrome-labelled, specific anti-lectin antibodies.Galanthus nivalis (snowdrop), NPA from Narcissus pseudonarcissus (daffodil) and APA from Allium porrum (leek). In addition, two targets for these Man-specific lectins in the replication cycle of SARS-CoV have been identified, one in the early phase of the replication cycle during viral attachment, and a second target at the end of the infection cycle [Lablab purpureus), which specifically recognizes N-glycans of the complex type occurring on the surface of coronavirus envelope, was demonstrated to neutralize SARS-CoV-2 and prevent both viral protein production and cytopathic effects in host (mice) cells [The antiviral properties of Man-specific seaweed lectins and the application of these lectins as blocking agents for the replication of enveloped viruses still requires more investigation. So far, the antiviral properties of Man-specific seaweed lectins, have been demonstrated essentially in in vitro conditions . Indeed,on cycle . More ree) cells .Hippeastrum hybridum, at the end of the SARS-CoV infection cycle [At the molecular level, the mechanism of action for Man-specific lectins is primarily referred to a masking effect of the molecular surface of S-glycoprotein RBDs due to their interaction with the Man-containing glycans, thus hampering the proper attachment of the virions to the host cell receptors and preventing the viral replication. However, the identification of a second target for HHA, the Man-specific lectin from on cycle , suggestAlthough lectins remain attractive anti-coronavirus candidates, at present it remains difficult to correctly assess the actual role of these natural compounds in the therapeutic armamentarium, to fight against SARS-CoV-2, the coronavirus responsible for the highly transmissible infectious COVID-19 ,105,106.


Correction to: IMA Fungus (2019) 10:15https://doi.org/10.1186/s43008-019-0015-5Incorrect sequence:The published article (Zhang and Zhang ">NAD4L_NC_001715Correct sequence:ATGCTTTTAGAAATAATAACAGCTTATAAAATAGGAACAATCTTATTTTTAATTGGAATTTTAGGTTTCATTATCAATAGACAAAATATTCTTTTACTTATTATCTCTATTGAAATGACTTTATTAGCTATTAGTTTTATTATTATTTGTTCTGCTCTTTTCCTTGATGATTCTGCAGCAGCTTGTTTTTCACTTTATATTTTAGCTCTTGCTGGTTCAGAAGCTGCAATTGGTCTTTCACTTTTAGTTTTATTCCATAGATTTAGAGGATCAGTATTAATTTCAGCTTCTCGACAATAG"">nad4L_NC_036382ATGAGTTTAACTTTAGTACTTTTTTTAATAGGAATCTTAGGATTCGTATTTAATAGAAAAAATATAATATTAATGCTTATTTCTATAGAAATAATGCTATTATCTATAACATTTTTAATATTGGTAAGTTCTATTAATCTTGACGATATAATAGGACAAACATATGCTATATACATTATAGTAGTTGCTGGTGCAGAATCTGCTATCGGTTTAGCTATTTTAGTAGCTTTTTATAGACTAAGAGGAAGTATCGCAATAGAATATAAATAA"To be in concordance with this change, “P263” in the nad4L column should be changed to “P239” in Table 3.The authors would like to apologise for any inconvenience caused.


Bacillus species, B. CMC1) and a regulator molecule  by a vapor-phased encapsulation method with simple steps of water sublimation and poly-p-xylylene deposition in chemical vapor deposition (CVD) process. Mechanically, the capsule construct exhibited a controllable shape and dimensions, and was composed of highly biocompatible poly-p-xylylene as the matrix with homogeneously distributed bacteria and CMC molecules. Versatility of the encapsulation of the molecules at the desired concentrations was achieved in the vapor-phased sublimation and deposition fabrication process. The discovery of the fabricated capsule revealed that viable living B. CMC1 inhabited the capsule, and the capsule enhanced bacterial growth due to the materials and process used. Biologically, the encapsulated B. CMC1 demonstrated viable and functional enzyme activity for cellulase activation, and such activity was regulatable and proportional to the concentration of the decorated CMC molecules in the same capsule construct. Impressively, 13% of cellulase activity increase was realized by encapsulation of B. CMC1 by poly-p-xylylene, and a further 34% of cellulase activity increase was achieved by encapsulation of additional 2.5% CMC. Accordingly, this synergistic effectiveness of the capsule constructs was established by combining enzymatic B. CMC1 bacteria and its regulatory CMC by poly-p-xylylene encapsulation process. This reported encapsulation process exhibited other advantages, including the use of simple steps and a dry and clean process free of harmful chemicals; most importantly, the process is scalable for mass production. The present study represents a novel method to fabricate bacteria-encapsulated capsule for cellulose degradation in bioremediation that can be used in various applications, such as wastewater treatment and transforming of cellulose into glucose for biofuel production. Moreover, the concept of this vapor-phased encapsulation technology can be correspondingly used to encapsulate multiple bacteria and regulators to enhance the specific enzyme functions for degradation of various organic matters.A regulatable bioremediation capsule material was synthesized with isolated single-strain bacteria ( Bioremediation provides promising solutions for the removal of environmental pollutants, toxic elements, and poisoning management for clinical purposes ,2,3. Varp-xylylenes of United States Pharmacopeia (USP) class VI with high biocompatibility and chemical resistance to strong acids, bases, and solvents was used for encapsulation during the fabrication process. The fabrication is performed in one step with a dry and clean vapor phase, which is desirable for sensitive biological substances such as cells, enzymes, growth factors, and other functional peptides and proteins [p-xylylene polymer on a template substrate that is eventually sublimated, and by manipulating the mass transport during the processing conditions, diverse species with distinct thermodynamic properties were subjected to sublimation and deposition within the confined space of the templates. Finally, transformation of the template resulted in the construction of composite materials with defined physical properties in terms of porosity, bulk size and geometry, and chemical functionality by the compartmentalized functional entities and the devised interfacial chemistries [Bacillus species CMC1 (hereafter referred to as B. CMC1) with specific enzymatic function and the same precision to localize and distribute B. CMC1 due to the encapsulation technique, enabling well-controlled bacterial growth activities and functions, and (ii) a regulator molecule, carboxymethyl cellulose (CMC), with a customizable encapsulation concentration to provide the same customizable stimulation dosages to regulate the enzymatic functions of the neighboring B. CMC1 bacteria. The fabricated and encapsulated capsules were composed of inhabitant bacteria from (i) and the surrounding conditioning regulator molecules from (ii), and the synergistic activities exhibited by (i) and (ii) were able to deliver a combination of controlled enzymatic factors for bioremediation in the devised microenvironments , 0.2 g CaCl2 , 0.2 g KNO2 , 2 g NH4NO3 , 0.3 g Na2HPO4 , 5 g glucose , and 2.5 mL tween 80  were added and then pH with NaOH to 7.0. The genomic DNA of the isolated bacterial strain was extracted using a commercial genomic DNA extraction kit . Then, the sequence of its 16S rRNA was confirmed by polymerase chain reaction by using the primers F8 (5′GAGAGTTTGATCCTGGCTCAG3′) and R1492 (5′GGTTACCTTGTTACGACTT3′). The obtained 16S rRNA sequence from the isolated bacteria wasWastewater and activated sludge were collected from the sewage of a pig farm in Taoyuan, Taiwan. All tools and reservoirs were sterilized before sampling. Activated sludge was stored immediately at 4 °C after collection. The wastewater and activated sludge were used for further culturing of environmental bacteria in liquid nutrient medium. In one liter of culture medium, 3 g peptone , 1 g yeast extract , 5 g NaCl , 0.2 g MgSOTGCTATACATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGC.https://www.ezbiocloud.net/. The isolated bacteria were the Bacillus species with the top ranking of B. subtilis and was named Bacillus species CMC1 (B. CMC1) in the study.The sequence was further confirmed according to the 16S rRNA database at B. CMC1 was cultured for 30 h at 30 °C and then washed twice with diH2O by centrifugation and resuspended into final solutions , 1.5% CMC, or 2.5% CMC) to prepare samples with the same concentration of bacteria (~1 × 108 bacteria/mL) on the same day of capsule fabrication. The prepared bacterial solution with/without CMC was solidified by liquid nitrogen for later encapsulation. A home-built vapor deposition system was used for the encapsulation and fabrication process in this study , 0.2 g KCl , and 1 g MgSO4·7H2O  were added, and then the pH was adjusted to 6.3 with NaOH. Then, 9 g agar  and 10 mg trypan blue  were added. A single bacterial capsule (100 µL in volume) was seeded in the center of one 9 cm plate and left at 30 °C for up to 140 h for subsequent enzyme functioning zone quantification. For the quantification of DNA and RNA, a single bacterial capsule (100 µL in volume) was seeded in a 50 mL centrifuge tube with 30 mL of growth culture medium inside, and the culture was left at 30 °C for 50 h on an orbital shaker with a 100 rpm shaking speed. The same volume of the culture (5 mL) from encapsulated bacteria was centrifuged for genomic DNA extraction using a commercial extraction kit  for the following DNA quantification. The same number of bacterial cells (2 × 108 cells) was used for total RNA extraction by using a commercial extraction kit  for total RNA quantification. The concentrations of DNA and RNA were measured by a NanoDrop Spectrophotometer . For this, 1 µL of purified DNA and RNA were used in the analysis. Note that only the DNA and RNA with the 260/280 ratio between 1.8 and 2.0 were used for concentration measurements, ensuring the sufficient purity of DNA and RNA for further quantification analysis.Cellulase activity was examined on 9 cm agar plates by quantifying the size of the enzyme functioning zones. To one liter of cellulase visualizing agar, 1 g yeast extract , 1 g NHB. CMC1 in the porous parylene structure. A VK-9510 3D profile microscope  was used to analyze the external architecture of the bacterial capsules and the existence of the bacteria inside the structure. SEM images were recorded using a NovaTM NanoSEM  operated at a primary energy of 10 keV and a pressure of 5 × 10−6 Torr to detailed the internal structure of the bacterial capsule. EDS elemental point analyses were captured for the quantification of the studied elements. 3D analysis of the interior structure was performed by using Bruker Skyscan 2211  at 2.0 μm/pixel resolution. The setting of the voltage was 40 kVp, whereas the current was 700 μA at 8 Watt output with microfocus mode. Image reconstruction, ring artifacts, and beam-hardening correction were performed using reconstruction software, Instarecon . FT-IR spectra of the fabricated capsules were recorded by a Spectrum 100 spectrometer equipped with an ATR detector . The recorded spectra ranged from 600 to 4500 cm−1 with 4 scan times at 4 cm−1 resolution.A Nikon ECLIPSE 80i fluorescence microscope  with a visible light source was used to visualize the crystal violet-stained p-xylylene in one continuous step [p-xylylene molecules to replace the resulting space when the sublimated ice/water molecules evaporated from the ice templates. The resultant construct consisted of a three-dimensional porous poly-p-xylylene matrix with encapsulated B. CMC1 bacteria in the matrix , and confocal microscopy to verify the localization and distribution of the bacteria . The imaructures a. The anB. CMC1, we hypothesized that the bacteria were resistant to low temperatures during iced template preparation and low-pressure conditions (approximately 10−3 Torr) during the sublimation and deposition process. Cultured samples of encapsulated B. CMC1 were compared to samples of B. CMC1 without encapsulation (positive control), and their cell densities were monitored by OD600 measurement. The results showed comparable bacterial growth patterns and activities in liquid medium for both types of bacteria during the culture time frame of 10 h  various concentrations of CMC . Subsequently, the same sublimation and deposition process was performed to resolve the capsule construct that comprised a poly-p-xylylene matrix with simultaneous encapsulation (i) and (ii) the same matrix structure. In addition to the already confirmed encapsulation of B. CMC1, the use of FT-IR analysis indicated that the intensity of the characteristic –C=O and –CH–O–CH2 peaks from CMC observed at 1558 and 1050 cm−1, respectively, gradually increased from a low concentration to higher concentrations of CMC content in the fabricated capsules  from poly-p-xylylene, additionally showed consistency by stoichiometry of the encapsulated CMC concentrations  exhibited approximately 10 h of bacterial growth in the lag phase and showed consistency with the samples with only B. CMC1 encapsulation  were studied in parallel to bare samples with unregulated B. CMC1 (0% CMC), and their enzymatic cellulase activities were first examined on agar plate results for up to 140 h  to a 0.25% regulation was found, and an anticipated enhancement of the expression with increasing CMC regulation compositions was accordingly discovered with a 13.1% increase for 1.5% CMC and 27.4% for 2.5% CMC of the studied capsule samples. On the other hand, based on the same bacteria number to extract their total RNA, the results in B. CMC1 was regulated for the synergistic capsules, and the synergy was found to be consistent with the same regulatable and enhancement of the bacterial population in the aforementioned study. Collectively, the results unambiguously verified the hypothesis that synergic effectiveness was achieved by vapor encapsulation of (i) B. CMC1 and (ii) the CMC regulator in the same fabricated capsule constructs, and the regulation was achievable through the versatile use of the encapsulated CMC compositions.More evidence showing the enhancement of the enzyme activities was further verified through the analysis of the genomic information, including the amount of genomic DNA and total RNA from the cultured samples of the capsules at a time point of 50 h, which was in the early stage of stationary phase in the bacterial growth curve. Calculation and quantification of the genomic DNA was performed based on the same volume of bacteria obtained from the capsule samples, and as shown in B. CMC1 was achieved in the current study. Because of the versatility of various types of remedial bacteria and regulator molecules, unlimited applications are expected beyond those shown in the report. In addition, the use of water/ice templates and a dry and clean vapor-phased process preserved the sensitive biomolecules and their biological functions, and the final fabricated capsule construct was composed of a USP Class VI compatible poly-p-xylylene matrix. Due to this specific production process, this novel fabrication process can not only encapsulate functional bacteria, regulating molecules, but also other potential absorbing material to concentrate the organic matters for degradation. It was reported that the encapsulation of phenol metabolic bacteria in microfiltration membrane capsules alone can create a confined environment to enhance the bioremediation of efficiency of encapsulated bacteria [p-xylylene fabrication technology can constitute a defined inner porous structure to capture different size of organic particles to further concentrate organic matters and a controllable size and shape of the products to fit to different situations. Until now, most of the present studies still focused on identifying the bacteria with specific enzyme functions to use in the bioremediation process [Synergistic and regulatable bioremediation of cellulose by encapsulated bacteria . The pol process ,25,26. W


Scientific Reports 10.1038/s41598-020-68172-2, published online 09 July 2020Correction to: The Supplementary Information file that accompanies this Article contains errors in Supplemental Table 1.her9 F, “CCTGACGGAGAACTGAACACAAGACACACA”The primer sequence reported for should read:“CGCCACACACACGCTCGTGT”her9 R, “TTTCTCAATGGTACGGCGGGTGCTCTGGGC”The sequence reported for should read:“CGGCTTGTGGAACGCCCGAA”her9 CR F, “AAGCTTCCTGACGGAGAACTGAACACAAGACACACA”The sequence reported for should read:“AAGCTTTGGCTTCTACCGGACTCAACTTTGGTGTTT”her9 CR R, “GAATTCTTTCTCAATGGTACGGCGGGTGCTCTGGGC”Finally, the sequence reported for should read:“GAATTCCCATGAAAACTTTATAAGTTCATATGAGATGCGC”


Recent approval of chimeric antigen receptor (CAR) T cell therapy by the European Medicines Agency (EMA)/Federal and Drug Administration (FDA) and the remarkable results of CAR T clinical trials illustrate the curative potential of this therapy. While CARs against a multitude of different antigens are being developed and tested (pre)clinically, there is still a need for optimization. The use of single-chain variable fragments (scFvs) as targeting moieties hampers the quick generation of functional CARs and could potentially limit the efficacy. Instead, nanobodies may largely circumvent these difficulties. We used an available nanobody library generated after immunization of llamas against Cluster of Differentiation (CD) 20 through DNA vaccination or against the ectodomain of CD33 using soluble protein. The nanobody specific sequences were amplified by PCR and cloned by Gibson Assembly into a retroviral vector containing two different second-generation CAR constructs. After transduction in T cells, we observed high cell membrane nanoCAR expression in all cases. Following stimulation of nanoCAR-expressing T cells with antigen-positive cell lines, robust T cell activation, cytokine production and tumor cell lysis both in vitro and in vivo was observed. The use of nanobody technology in combination with PCR and Gibson Assembly allows for the rapid and effective generation of compact CARs.  CARs are synthetic chimeric receptors consisting of an antibody based extracellular part to recognize specific antigens expressed on the surface of tumor cells and an intracellular part containing (co)stimulatory signals derived from CD3ζ, CD28 and/or 4_1BB ,10,11. CH) and variable light (VL) regions and the generation of a single-chain variable fragment (scFv) for target recognition [H and VL of the different scFvs [CARs that obtained market authorization and those that are clinically tested rely on the generation of a monoclonal antibody, the sequencing of the variable heavy  found in Camelidae [H gene family III and their small size [Nanobodies can potentially be an alternative antigen-binding moiety. Nanobodies, first described by Hamers-Castermans, are isolated from the Vamelidae . The strall size ,29. No oall size ,31,32,33all size . Finallyall size . Therefoall size . Both siall size ,40,41,42all size .We previously described the use of a dual specific CAR based on nanobodies . To expa1 CH2CH3 (Fc) spacer, the CD28 transmembrane domain and the CD28 and CD3ζ intracellular signaling domains. The 4_1BB:ζ construct contains the CD8α hinge and transmembrane region followed by the 4_1BB and CD3ζ intracellular signaling moieties. Both CAR backbone constructs were cloned in a retroviral plasmid that contains an IRES-eGFP sequence to allow easy detection of CAR-expressing cells. At the 5′ position we incorporated a BamHI restriction site . The nanobody specific sequences were amplified by PCR using primers with 15–20 nucleotide overhangs complementary to the 5′ retroviral plasmid (forward primer) or the 3′ CAR sequence (reverse primer). The forward primer also encoded a leader sequence derived from the L-kappa murine leader sequence. The PCR products were purified, and Gibson Assembly was used to introduce the amplified sequence into the CAR vector . The nanobody sequences specific for human CD33 were derived from a llama nanobody phage library. This library was constructed from peripheral blood lymphocytes obtained after immunization of a llama with the extracellular domain of human CD33 protein. Three different nanobody clones were randomly selected and amplified by PCR, purified and cloned into the CAR backbone by Gibson Assembly, resulting in three CD33-specific nanoCAR constructs. After sequence confirmation, we produced retroviral particles and subsequently transduced peripheral blood mononuclear cells (PBMC). NanoCAR expression was confirmed by flow cytometry using an antibody specific for the nanobody protein . Transdu− ovarian cancer cell line SKOV3 was used as negative control. Most acute myeloid leukemia cells express CD33. A panel of AML cell lines was analyzed for CD33 expression A. Flow c+ cell lines. The cytotoxicity was driven by CAR binding to cognate antigens, since CD33+ cell lines were neglected by non-transduced T cells and CD33− SKOV3 cells did not elicit target cell lysis. Next, we evaluated cytokine production of our CD33-specific nanoCAR T cells 5 h after co-incubation with our target cell panel by intracellular staining for interferon-γ (IFN-γ) and interleukin-2 (IL-2). The nanoCAR-expressing cells were able to produce both IFN-γ and IL-2 after incubation with CD33+ MOLM13 cells. Incubation with the CD33− cell line SKOV3 resulted in no significant expression of IL-2 or IFN-γ  mice were intravenously injected with Thp1 cells expressing firefly luciferase. The cells were allowed to engraft and expand. After checking engraftment 7 days after injection, a single tail vein injection of nanoCAR T cells was administered day 0 in A. Follow+ hematopoietic precursor cells (HPC) were isolated from different cord blood donors and analyzed for CD33 expression. Only CD34dimCD38dim HPC expressed CD33 although at a lower level compared with leukemic cell lines  through their CH2 domain. Earlier studies of the IgG1 Fc spacer showed off-target CAR T cell activation by FcRγ+ myeloid and lymphoid cells and speculated that potential activation-induced cell death (AICD) could occur [We also noticed no survival benefit nor disease control in NSG mice injected with CD33-1-CD28:ζ nanoCAR T cells. Furthermore, we were not able to detect any eGFPld occur . Other gld occur ,55,56,57In summary, we have shown that our technique of using nanobodies, PCR and Gibson Assembly is a rapid and efficient way to generate nanoCAR T cells with a 100% success rate for the six randomly selected nanobody clones. We chose two completely different antigens: CD20, a tetraspanner and CD33, a single pass receptor, to test our technique. We strongly believe that the use of nanobodies is advantageous over the use of scFvs, since nanobodies are monomeric structures that (i) will probably not aggregate on the T cell surface and therefore not induce premature T cell activation and exhaustion ; (ii) wiProcedures for immunization of llamas, preparation of mRNA, construction of the library, and panning were performed as previously described .l-glutamine , 100 IU/mL penicillin  and 100 IU/mL streptomycin  . SKOV3 was cultured in DMEM  supplemented with 10% fetal calf serum , 2 mM L-glutamine , 100 IU/mL penicillin  and 100 IU/mL streptomycin . Thp1 was cultured in RPMI  supplemented with 0.05 mM β-mercaptoethanol, 10% fetal calf serum , 2mM L-glutamine , 100 IU/mL penicillin  and 100 IU/mL streptomycin .All the cell lines were cultured as per American Type Culture Collection  recommendations. RL, Raji, MOLM13, U937, were cultured in RPMI , supplemented with 10% fetal calf serum , 2 mM L-glutamine , 100 IU/mL penicillin  and 100 IU/mL streptomycin . Jurkat and HL60 were cultured in IMDM  supplemented with 10 % fetal calf serum , 2 mM Escherichia coli (High Efficiency), NEB) and plated on agar. After overnight incubation, colonies were selected and grown in liquid lysogenic broth  overnight. Plasmids were isolated and sequenced. Colonies containing the correct plasmid were further cultured and midipreps (Qiagen) were performed. The different constructs, as shown in 5 cells per 6 cm dish  and placed overnight in a 7% CO2 incubator at 37 °C. Next day, the plasmids encoding the nanoCAR constructs were transfected using calcium phosphate as follows: per 6 cm dish, 10 µg plasmid DNA was diluted in 36 µL 2M CaCl2 (homemade) and subsequently nuclease free water  was added to a total volume of 300 µL. The DNA-CaCl2 solution was pipetted dropwise into a 15 mL polystyrene tube containing 300 µL 2× HEPES-buffered saline (HBS) solution (homemade) while blowing air bubbles in the 2× HBS buffer. The mixture was incubated for 15 min at room temperature and then added dropwise to the cells. Ten minutes before the addition of the DNA mixture, 2 mL medium was added to the cells containing 1 µL of a 200 mM chloroquine  solution. Cells were placed in a 7% CO2 incubator at 37 °C overnight. Medium was refreshed at day one. At day two, cells were analyzed for transfection efficiency and transgene expression and reseeded in a T75 culture flask  in selection medium containing 2 µg/mL puromycin (Sigma). After an additional two days, selection medium was replaced by medium without puromycin. At day six and day 10, the selection cycle was repeated. Finally, at day fourteen, retroviral supernatant was collected and was frozen at −80 °C until use. Phoenix A cells were analyzed for nanoCAR and eGFP expression at days of reseeding and at day fourteen. Viral particles were produced using the Phoenix A packaging cell line. Phoenix A cells were seeded at 7.5 × 10+ cells was determined by flow cytometry and T cells were stimulated with Immunocult Human CD3/CD28/CD2 T cell activator  per fabricator instructions in cIMDM, in the presence of 10 ng/mL IL-12 . Cells were harvested 72 h after stimulation and resuspended in retroviral supernatant and centrifuged for 90 min at 1000× g at 32 °C on retronectin  coated plates. Buffy coats from healthy donors were obtained from the Belgian Red Cross and used following the guidelines of the Medical Ethical Committee of Ghent University Hospital, after informed consent had been obtained, in accordance with the Declaration of Helsinki. PBMC were isolated by Lymphrop  gradient centrifugation. The percentage of CD3Transduced cells were detected by eGFP expression and after staining with a nanbody specific antibody. Transduced cells were sorted and expanded on irradiated allogenic feeder cells, consisting of a mixture of 40 Gy irradiated peripheral blood mononuclear cells and 50 Gy irradiated JY cells. Cells were cultured in cIMDM, supplemented with 1 μg/mL phytohemagglutinin  (Roche) was added on day five and day ten. Cells were restimulated every seven–fourteen days.Staining of surface markers was performed as described earlier . The fol+, Raji, RL and SKOV3. Cytotoxicity assay was performed as previously described . The folDetection of cytokine producing cells was performed as previously described , with th4 Thp1 cells) in cIMDM. Cells were stained with CD3, CD4 and CD8α at the start of the co-culture and at day three, seven, ten and fourteen. At day seven of co-culture, 2 × 104 Thp1 cells were added to the remaining wells. Cell numbers were determined by flow cytometry. T cells expressing nanoCARs were incubated with Thp1 cells at an effector/target ratio of 0.025:1  was performed after intraperitoneal injection of 150 mg/kg bodyweight d-luciferin (Perkin Elmer) and 5 × 106 nanoCAR T cells were intravenously injected. Tumor progression was followed up by IVIS imaging. Mice were checked for overall health status and scarified when humane endpoints were reached. NSG mice were injected intravenously with 2 × 106 RL cells were subcutaneously injected. The cells were allowed to form a solid mass and at day eighteen, 5 × 106 nanoCAR T cells were intravenously injected. Tumor progression was followed up by caliper. Mice were checked for overall health status and scarified when human endpoints were reached. For the RL model, 2 × 10+ HPC were purified using CD34 MicroBead Kit (MACS Miltenyi Biotech) per manufactures instructions. T cells expressing the nanoCARs were cultured with CD34+ HPC in cIMDM supplemented with stem cell factor , thrombopoietin  and FMS-like tyrosine kinase 3 ligand , all three at 100 ng/mL with PBMC at a 1:1 ratio. At the start and at 24 h, 48 h and at 72 h of the co-culture, cells were harvest and stained for CD3, CD34, CD38 and CD33. Subsequently, cells were analyzed by flow cytometry and absolute cell counts were determined.Cord blood was acquired through the Belgian Red Cross and used following the guidelines of the Medical Ethical Committee of Ghent University Hospital (EC/UZG 2015/0768), after informed consent had been obtained, in accordance with the Declaration of Helsinki. PBMC were isolated using density gradient centrifugation. CD34Underlined sequences indicate overlap with the LZRS vector, cursive sequences indicate BamHI site, sequences in bold indicate leader sequence, sequences in upper case indicate overlap with nanobody sequences and sequences in upper case and bold indicate overlap with CAR backbone.Nanobody specific sequence amplification.Fw primer:ggatccgggtggaccatcctctagactgccgccatggattttcaggtgcagattttcagcttcctgctaatcagtgcctcagtcataatgtctagaCAGGTGCAGCTGCAGGAG3′5′Rev primer 4_1BB:ζ backbone:ggatccACTGAGGAGACGGTGACCTG3′5′GGTCGCGGCGCTGGCGTCGTGGTCRev primer CD28:ζ backbone:ggatccACTGAGGAGACGGTGACCTG3′5′AGGAGATTTGGGCTCGGCGGGCDNA sequence of CD28:ζ CAR backbone construct:gggtggaccatcctctagactgccggatccgccggatccCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCTTCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAAAGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGATAAatggattttcaggtgcagattttcagcttcctgctaatcagtgcctcagtcataatgtctagaGTDNA sequence of 4_1BB:ζ CAR backbone construct:gggtggaccatcctctagactgccggatccgccggatccGACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAAatggattttcaggtgcagattttcagcttcctgctaatcagtgcctcagtcataatgtctagaGTData were analyzed with GraphPad Prism Software . Statistical differences between groups or conditions were determined by two-way ANOVA, followed by Bonferroni post hoc test. Survival curves were compared using the log-rank Mantel–Cox test.


Ppt1, in cancer cells reduced priming and cytotoxic capacity of primed T cells. Exposure of antigen-primed T cells to macrophage-conditioned medium derived from macrophages treated with PPT1 inhibitors enhanced melanoma-specific killing. Genetic or chemical Ppt1 inhibition resulted in M2 to M1 phenotype switching in macrophages. The combination was associated with a reduction in myeloid-derived suppressor cells in the tumor. Ppt1 inhibition by HCQ, or DC661, induced cyclic GMP-AMP synthase/stimulator of interferon genes/TANK binding kinase 1 pathway activation and the secretion of interferon-β in macrophages, the latter being a key component for augmented T cell–mediated cytotoxicity. Genetic Ppt1 inhibition produced similar findings. These data provide the rationale for this combination in melanoma clinical trials and further investigation in other cancers.New strategies are needed to enhance the efficacy of anti–programmed cell death protein antibody (anti–PD-1 Ab) in cancer. Here, we report that inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of chloroquine derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti–PD-1 Ab in melanoma. The combination resulted in tumor growth impairment and improved survival in mouse models. Genetic suppression of core autophagy genes, but not   Inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of CQ derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti-PD-1 Ab in murine melanoma models. While there have been extensive efforts to combine other T cell–stimulating factors with anti–programmed cell death protein antibody anti–PD-1 Ab), there is an increasing interest in identifying T cell–independent strategies that will augment the efficacy of anti–PD-1 Ab. Tumor cell autophagy has been identified as a major resistance mechanism to targeted therapy and chemotherapy. Chloroquine and hydroxychloroquine (CQ and HCQ) are the only drugs that have been tested as autophagy inhibitors in clinical trials in patients with cancer. However, there are conflicting reports about whether as a single agent CQ derivatives augment, impair, or have no effects on antitumor immunity  Ab, ther.Ppt1 (siPpt1) were all able to convert M2 to M1 tumor-associated macrophages (TAMs). Conditioned media from macrophages treated with Ppt1 inhibitors were able to enhance T cell–mediated cancer cell killing. Interestingly, the combination of HCQ and anti–PD-1 Ab resulted in a change in TAM polarization and a significant reduction in myeloid-derived suppressor cell (MDSC) infiltration in vivo while each single agent did not. The mechanism by which PPT1 inhibitors produced macrophage phenotype switching was dependent on mitochondrial calcium release and p38 activation. PPT1 inhibition also induced the cyclic GMP-AMP synthase/stimulator of interferon genes/TANK binding kinase 1 (cGAS/STING/TBK1) pathway to induce IFN-β release from macrophages. PPt1 inhibition–induced IFN-β release was critical for the augmentation of antigen-primed T cell killing of melanoma cells. These data suggest that this combination, which can be immediately tested in the clinic, could provide an alternative rational combination approach for melanoma immunotherapy.Recently, we identified the major molecular target of CQ derivatives as the lysosomal protein palmitoyl-protein thioesterase 1 (PPT1) , 4. Meanhttps://doi.org/10.1172/jci.insight.133225DS1). Tumors harvested at the end of the experiment were significantly smaller with combination compared with monotherapy arms , and Atg7. KD of these 3 genes in B16 cells produced reduced autophagic flux as evidenced by increase in p62. While Lc3b-II levels did not change much with siUlk1 or siPik3c3 as is often observed  to an antitumorigenic M1) phenotype or mouse macrophages in vitro and in vivo  phenotyp. Morpholhenotype . KD of crization . Ppt1 KD+ T cells play a major role in the elimination of tumor cells and regression of tumors by their cytotoxic activity  activates Cre recombinase, and melanocytes express mutant Braf and lose Pten. Spontaneous melanoma tumors arise on the skin of these mice with 100% penetrance. In concordance with the B16 tumor model, we found that HCQ significantly enhanced the antitumor response of anti–PD-1 Ab  (+CD11b+Ly6C+Ly6G–CX3CR1+ monocytes and spleen eosinophils (CD45+CD11b+Siglec F+) and no significant change in DCs (CD45+CD11b+CD11c+) and PMN-MDSCs (CD45+CD11b+Ly6CloLy6Ghi)  T cells compartment in the spleen and tumor  . There woLy6Ghi) . There wnd tumor . Taken tPpt1 also resulted in increased levels of p-p38 compared with siNon-target control  . HCQ, DC control . PPT1 in control . Inhibit control .+/Ca2+ exchanger, completely abrogated the increased p38 phosphorylation induced by lysosomal inhibitors , while a comparison of DC661 versus control showed 47 significantly changed proteins out of a total of 3166 proteins identified with high confidence . MCM was subjected to proteome analysis. Comparison of HCQ versus control showed 64 significantly changed proteins  CAGCATCTTCTTGGCAGACATAAATCAAGAGAGGTGTGTCAATGAGTCCTACAAGAAGAACCTGATGGCCCTCAAGAAGTTTGTGATGGTGAAATTCTTTAATGATTCCATTGTGGACCCTGTCGACTCTGAGTGGTTTGGATTTTACAGAAGTGGCCAAGCTAAGGAAACCATTCCCCTCCAGGAGAGCACTCTATACACAGAGGACCGCCTGGGGCTAAAGAAAATGGACAAAGCAGGAAAGCTAGTGTTTCTGGCTAAGGAAGGGGACCATCTTCAAATATCTAAAGAATGGTTTACTGCCCACATCATACCTTTTCTTAAGTGATGCCCTGGCACTTTATAGCAGAGTTCATGAAACCACAGCTCTTCCAAGCCATGTACATAGTTCATGCTCAGCCTGAACTCTAATCTAGCCTGCAACCAGCCCTTCTCTCCTCTTATCATCTAACATACCCTACTTGGAAAGATCTAAGATCTCAATCTTATCCTTTGCCGCCT. PpT1 siRNA pool 2 (Santa Cruz Biotechnology sc-142398) AGGCGGCAAAGGATAAGATTGAGATCTTAGATCTTTCCAAGTAGGGTATGTTAGATGATAAGAGGAGAGAAGGGCTGGTTGCAGGCTAGATTAGAGTTCAGGCTGAGCATGAACTATGTACATGGCTTGGAAGAGCTGTGGTTTCATGAACTCTGCTATAAAGTGCCAGGGCATCACTTAAGAAAAGGTATGATGTGGGCAGTAAACCATTCTTTAGATATTTGAAGATGGTCCCCTTCCTTAGCCAGAAACACTAGCTTTCCTGCTTTGTCCATTTTCTTTAGCCCCAGGCGGTCCTCTGTGTATAGAGTGCTCTCCTGGAGGGGAATGGTTTCCTTAGCTTGGCCACTTCTGTAAAATCCAAACCACTCAGAGTCGACAGGGTCCACAATGGAATCATTAAAGAATTTCACCATCACAAACTTCTTGAGGGCCATCAGGTTCTTCTTGTAGGACTCATTGACACACCTCTCTTGATTTATGTCTGCCAAGAAGATGCTG. Ppt1 siRNA single duplex  CTGGTTGCAGGCTAGATTAGAGTTCAGGCTGAGCATGAACTATGTACATGGCTTGGAAGAGCTGTGGTTTCATGAACTCTGCTATAAAGTGCCAGGGCATCACTTAAGAAAAGGTATGATGTGGGCAGTAAACCATTCTTTAGATATTTGAAGATGGTCCCCTTCCTTAGCCAGAAACACTAGCTTTCCTGCTTTGTCCATTTTCTTTAGCCCCAGGCGGTCCTCTGTGTATAGAGTGCTCTCCTGGAGGGGAATGGTTTCCTTAGCTTGGCCACTTCTGTAAAATCCAAACCACTCAGAGTCGACAGGGTCCACAATGGAATCATTAAAGAATTTCACCATCACAAACTT. Fluo-4, AM, was used to stain the cells for calcium as per the manufacturer’s instructions .DC661 was provided in-house. Purity of the sample was determined by NMR spectroscopy and liquid chromatography-mass spectrometry. Mouse melanoma B16-F10 (CRL-6475) and RAW 264.7 (TIB-71) were purchased from ATCC. Cell lines were tested for mycoplasma biannually and authenticated using short-tandem repeat fingerprinting. Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 4.5 g/L glucose, sodium pyruvate, phenol red, and 10% fetal calf serum . BMDMs were isolated as described previously .Total RNA was isolated by RNA isolation kit  according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized using iScript Reverse Transcriptase kit with 500 ng of purified RNA as per manufacturer’s protocol . The qPCR reaction was set up using SYBR Green PCR Master Mix  containing 1 μL of cDNA. All measurements were carried out in duplicate, and Hsp90 was used as internal standard for ΔCT calculations. Gene expression analysis was done using the following primers: iNos forward (AGGAGGAGAGAGATCCGATTTAG), iNos reverse (TCAGACTTCCCTGTCTCAGTAG); Retnla/Fizz1 forward (TGCCAATCCAGCTAACTATCC), Retnla/Fizz1 reverse (GCAAAGCCACAAGCACAC); Hsp90 forward (GGGAGCTCATCTCCAATTCATC), Hsp90 reverse (GTCCTGTTTGCTGGGAATGA); Arg1 forward (TACCTGCTGGGAAGGAAGAA), and Arg1 reverse (CTGTAAGATAGGCCTCCCAGA).Atg7 gRNA AACTCCAACGTCAAGCGGGT sequences for targeting mouse cells were used as described previously (XhoI and NheI sites of pLX-sgRNA followed by restriction digest and ligation. Sanger sequencing was used to confirm that the Atg7 gRNAs and nontargeting control RNA were correctly subcloned in the pLX-sgRNA vector. B16 cells were first transfected with the pCW-Cas9 vector and selected with puromycin 4 μg/mL for 6 days and then transfected with the pLX-sgRNA containing the Atg7 gRNA and nontargeting gRNA using Lipofectamine 3000 (Thermo Fisher Scientific) based on the manufacturer’s instructions. After 48 hours’ incubation, growth medium was changed to selection medium containing blasticidin  6 μg/mL. After blasticidin selection for 12 days, the cells were treated with doxycycline 0.5–1 μg/mL to express Cas9 and induce Atg7 deletion. Doxycycline was replenished every 2 days for 2 weeks, after which the cells were harvested and analyzed by immunoblotting for Atg7 deletion (The nontargeting guide RNA (gRNA) TAGCGAACGTGTCCGGCGT and eviously . The 2 seviously . Subclondeletion .Whole-cell lysates and lysosomal extracts were immunoblotted as previously described , 6.5) with an equal volume of Matrigel  in the right flank of C57BL6/J mice. Daily injection of HCQ (60 mg/kg) and every-other-day injection of anti–PD-1 Ab (200 μg) commenced at the tumor size of 50 mm3. CA PtenloxP Tyr::CreERT2BRaf mice (The Jackson Laboratory) were treated topically on the back with 4-HT to elicit V600EBRaf and to silence Pten expression. Tumors were measured using electric calipers . Tumor volume was calculated as L × W2 × 0.5. In all animal experiments 2-tailed t test or 2-tailed t test for unequal variance was used to test the hypothesis that the addition of HCQ to anti–PD-1 Ab is significantly different compared with anti–PD-1 Ab + Veh.Tumor generation, measurement, and harvesting were performed as previously described , 9. BrieTumor was excised and digested with a tumor digestion kit  according to the manufacturer’s protocol.6) from the digested tumor were stained with antibodies (please see Cells (1 × 105) were cultured and irradiated with 25 Gy x-rays. Splenocytes were then cultured with (primed) or without (unprimed) irradiated B16 cells in the presence of IL-2 (5 IU/mL) and cocultured for 48 hours. Splenocytes cultured with concanavalin A (10 μg/mL) were used as a nonspecific T cell priming control. Priming was confirmed by IFN-γ ELISA of the supernatant. Primed splenocytes were then cocultured with freshly cultured B16 cells with various target (B16) to effector (splenocytes) ratios. The B16 cell death–associated LDH release and then percentage cytotoxicity were measured according to the manufacturer’s protocol .B16 tumor cells  to remove cellular debris. The supernatant was passed through a 0.2 μm filter, concentrated 50-fold using an Amicon Ultra 3K membrane, electrophoresed for 0.5 cm into an SDS-PAGE gel, and stained with colloidal Coomassie . The entire stained region was excised, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) coupled with a Nano-ACQUITY UPLC system using a 245-minute gradient and acquisition parameters as previously described  were computed for each mouse using linear regression of the natural logarithm of tumor volume on time; the estimated slopes for the HCQ and anti–PD-1 Ab or anti–PD-1 Ab + Veh groups were analyzed using a 2-sample t test. All t tests were 2 tailed. A P value less than 0.05 was considered significant. P values presented in the figures are for the test of the hypothesis that the expected mean when HCQ is added to anti–PD-1 Ab is significantly different from the expected mean for anti–PD-1 Ab + Veh. Adjusted P values are indicated as * when the adjusted P < 0.05 and as ^ when the adjusted P < 0.10. All analyses were done using SAS/STAT software, version 9.4 of the SAS system for Windows.For continuous variables, a 2-sample All animal experiments were performed in accordance with the protocols approved by the University of Pennsylvania Institutional Animal Care and Use Committee.GS, RO, ENO, EJW, DG, XX, DWS, PAG, and RKA conceived experiments. GS, RO, ENO, JM, JA, VWR, SL, SP, JLL, SH, AR, VJ, MCN, JDW designed the experiments, conducted the experiments, and collected the data. GS, XX, ENO, EJW, DG, JDW, DWS, PAG, and RKA supervised experiments and analyzed the data. GS, DWS, DG, PAG, and RKA wrote the manuscript with assistance from all the other authors.