The ligand-dependent suppression of TCR signaling by the immune checkpoint receptor LAG3 depends on the cytoplasmic RRFSALE motif.

Lymphocyte activation gene 3 (LAG3) is an inhibitory immune checkpoint receptor that restrains autoimmune and antitumor responses, but its evolutionarily conserved cytoplasmic tail lacks classical inhibitory motifs. Major histocompatibility complex class II (MHC class II) is an established LAG3 ligand, and fibrinogen-like protein 1 (FGL1), lymph node sinusoidal endothelial cell C-type lectin (LSECtin), and Galectin-3 have been proposed as alternative binding partners that play important roles in LAG3 function. Here, we used a fluorescent human T cell reporter system to study the function of LAG3. We found that LAG3 reduced the response to T cell receptor stimulation in the presence of MHC class II molecules to a lesser extent compared with the receptor programmed cell death protein 1. Analysis of deletion mutants demonstrated that the RRFSALE motif in the cytoplasmic tail of LAG3 was necessary and sufficient for LAG3-mediated inhibition. In this system, FGL1, but not LSECtin or Galectin-3, acted as a LAG3 ligand that weakly induced inhibition. LAG3-blocking antibodies attenuated LAG3-mediated inhibition in our reporter cells and enhanced reporter cell activation even in the absence of LAG3 ligands, indicating that they could potentially enhance T cell responses independently of their blocking effect.

The soluble cytoplasmic tail of CD45 regulates T-cell activation via TLR4 signaling.

The soluble cytoplasmic tail of CD45 (ct-CD45) is a cleavage fragment of CD45, that is generated during the activation of human phagocytes. Upon release to the extracellular space, ct-CD45 binds to human T cells and inhibits their activation in vitro. Here, we studied the potential role of TLR4 as a receptor for ct-CD45. Treatment of Jurkat TLR4/CD14 reporter cells with ct-CD45 induced the upregulation of the reporter gene NFκB-eGFP and could be blocked by inhibitors of TLR4 signaling. Conversely, ct-CD45 did not promote the NFκB-controlled eGFP induction in reporter cells expressing TLR1, TLR2, and TLR6 transgenes and did not lead to the activation of the transcription factors NFκB, AP-1, and NFAT in a Jurkat reporter cell line expressing endogenous TLR5. Moreover, ct-CD45 binds to recombinant TLR4 in an in vitro assay and this association was reduced in the presence of oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine. Blockade of TLR4 with mAb HTA125 partially reversed the ct-CD45-mediated inhibition of T-cell proliferation. Interestingly, targeting of TLR4 with mAb W7C11 also suppressed T-cell proliferation. In summary, the results of this study demonstrate that ct-CD45 acts via a noncanonical TLR4 activation pathway on T cells, which modulates TCR signaling.

A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands.

Toll-like receptors (TLRs) are primary pattern recognition receptors (PRRs), which recognize conserved microbial components. They play important roles in innate immunity but also in the initiation of adaptive immune responses. Impurities containing TLR ligands are a frequent problem in research but also for the production of therapeutics since TLR ligands can exert strong immunomodulatory properties even in minute amounts. Consequently, there is a need for sensitive tools to detect TLR ligands with high sensitivity and specificity. Here we describe the development of a platform based on a highly sensitive NF-κB::eGFP reporter Jurkat JE6-1 T cell line for the detection of TLR ligands. Ectopic expression of TLRs and their coreceptors and CRISPR/Cas9-mediated deletion of endogenously expressed TLRs was deployed to generate reporter cell lines selectively expressing functional human TLR2/1, TLR2/6, TLR4 or TLR5 complexes. Using well-defined agonists for the respective TLR complexes we could demonstrate high specificity and sensitivity of the individual reporter lines. The limit of detection for LPS was below 1 pg/mL and ligands for TLR2/1 (Pam3CSK4), TLR2/6 (Fsl-1) and TLR5 (flagellin) were detected at concentrations as low as 1.0 ng/mL, 0.2 ng/mL and 10 pg/mL, respectively. We showed that the JE6-1 TLR reporter cells have the utility to characterize different commercially available TLR ligands as well as more complex samples like bacterially expressed proteins or allergen extracts. Impurities in preparations of microbial compounds as well as the lack of specificity of detection systems can lead to erroneous results and currently there is no consensus regarding the involvement of TLRs in the recognition of several molecules with proposed immunostimulatory functions. This reporter system represents a highly suitable tool for the definition of structural requirements for agonists of distinct TLR complexes.

GoldenBac: a simple, highly efficient, and widely applicable system for construction of multi-gene expression vectors for use with the baculovirus expression vector system.

BACKGROUND: Recombinant protein production and purification of large protein complexes in eukaryotes requires efficient methods to generate multi-gene expression constructs, where each individual gene is under the control of its own promoter and terminator. Current methods are based either on serial rounds of combination of several vectors containing loxP sites via the Cre-lox technology, or on multiple rounds of gene combination via PCR or other methods. These methods are multi-step, have lower efficiencies than single gene cloning, and may require laborious processes to verify that all genes of interest are present in the final product. Here, we describe a rapid and simple Golden Gate-based system for the generation of multi-gene expression constructs compatible with baculovirus expression vector systems (BEVS) using either Tn7 transposition or KO1629-based homologous recombination, which we refer to as "GoldenBac". RESULTS: This method is based on the construction of a series of vectors containing a promoter-gene of interest-terminator cassette flanked by cleavage sites of the BsaI type IIS restriction enzyme. This series of vectors can be cut by BsaI to excise cassettes with unique overhangs. In the same reaction, the cassettes are then ligated in the correct sequence in a final destination vector to generate multi-gene expression constructs containing 2-15 genes. Individual expression constructs can therefore be combined into a single vector in a single reaction, with over 90% efficiency when combining up to 14 expression cassettes. We demonstrate successful construction and expression of three different co-expression systems, the proteosomal lid complex, the anaphase promoting complex/cyclosome (APC/C), and a series of constructs used to test the effect of chaperone co-expression on the solubility of the HOIP protein. CONCLUSIONS: This robust, single-step cloning system provides an easy-to-use method for generation of multi-gene expression constructs for both transposition and homologous recombination-based baculovirus systems, making this technology available across all laboratories using baculovirus expression systems. This highly efficient and simple method allows for rapid incorporation of multi-gene expression cloning into the standardized service portfolio of protein production facilities and can also easily be adopted by any laboratory for routine generation of multi-gene baculovirus constructs.

Vaccinia Virus Immunomodulator A46: A Lipid and Protein-Binding Scaffold for Sequestering Host TIR-Domain Proteins.

Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1-83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all ß-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of ß-sheets. The A46(1-83) structure itself is a ß-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N- and C-terminal domains and SAXS analysis of full-length protein A46(1-240), we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88.