Humanity has been facing cancer since the beginning of its existence, and the number of new cancer cases grows each year. Immunotherapies based on immune checkpoint inhibition have been intensively developed over the last decade and focus on the blockade of the co-inhibitory molecule complexes such as CTLA-4 with its ligands CD80 and CD86, PD-1 with PD-L1 and PD-L2, and many more. The investigation and development of new immune checkpoints inhibitors is necessary for better cancer treatment. One way of inhibitor evaluation is to assess them in cell-based assays. In this protocol, we focus on describing the cell-based reporter platform for PD-1/PD-L1 inhibitors assessment, which is based on measuring the expression of eGFP under the transcription factor NF-κB, responsible for transcriptional program required for T-cell activation and differentiation.

Antibodies that block PD-1 signaling, known as immune checkpoint inhibitors (ICIs), have revolutionized cancer treatment. Small molecule inhibitors of the PD-1/PD-L1 interaction could serve as promising alternatives to antibody-based ICIs, offering advantages including reduced cost, the option of oral administration, and their small size allows for deeper tissue penetration. Numerous such compounds have been described, but many have not been evaluated in well-defined cellular systems, and comparative studies are scarce. We tested eleven small molecule inhibitors using Jurkat-PD-1-reporter cells activated by stimulator cells expressing PD-L1. Additionally, their effects on T cell reporter activation and viability were evaluated. We found that ARB-272572, INCB086550, Evixapodlin, and PD-1/PD-L1 Inhibitor 3 completely reversed the inhibitory effects of PD-1. Except for PD-1/PD-L1 Inhibitor 3, these compounds blocked PD-1 inhibition with EC50 values in the low nanomolar range. Interestingly, seven PD-1 inhibitors failed to fully block PD-1 inhibition in our cellular assay. BMS-1166 and PD-L1-IN3 showed some blocking capacity but could not fully restore the activation of PD-1 reporter cells. AUNP-12, BMS1, BMS202, CA170, and PD-1/PD-L1-IN-9 were ineffective at reducing PD-1-mediated reporter inhibition. Moreover, our data indicate that adverse effects on T cell reporter activation compromise the activity of several of the tested compounds. In summary, our results revealed that the majority of small molecule PD-1/PD-L1 blockers possess a limited capacity to reverse PD-1 inhibition in a T cell reporter platform and highlight the importance of cellular assays to assess the therapeutic potential of drugs targeting the PD-1/PD-L1 axis.

Occasional complete responses to immune checkpoint inhibitor therapy demonstrate that acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) can be immune-sensitive when appropriately targeted. Here, we analyzed AML/MDS patients (n=14) treated with the anti-TIM3 sabatolimab and the hypomethylating agent decitabine in a phase Ib clinical trial (NCT03066648) using single-cell RNA and T cell receptor (TCR) sequencing (n=6) and functional co-culture assays. Unlike T cell-restricted CTLA4 and PD1, TIM3 was broadly expressed across natural killer (NK)-cell, myeloid-cell, and T-cell populations. Therapy induced expansion of cytotoxic NK-cell subsets and enhanced type I interferon signaling. Fewer than 1% of bone marrow CD8+ T cells displayed a canonical exhaustion phenotype, and treatment preferably expanded small CD8+ T-cell clones in responders. Responders exhibited greater expansion of cytotoxic CD4+ T cells and B cells, as exemplified by a patient with pre-existing CD4+ T-cell large granular lymphocyte leukemia (T-LGLL) achieving an outstanding complete response lasting 23 months. Over 20% of this patient's lymphocytes were T-LGLL cells expressing a TCR capable of recognizing autologous blasts. Overall, our results suggest that anti-TIM3 combined with decitabine engages a distinct mechanism of immune activation compared to anti-PD1 and anti-CTLA4, preferentially expanding NK-cell and CD4+ T-cell populations.

Recent clinical trials in AML combining immune checkpoint inhibitors (anti-PD1, anti-CTLA4) with hypomethylating agents have yielded only modest response rates. However, there has been exceptional responders achieving durable complete responses in all these trials. TIM3 is a checkpoint molecule expressed both on immune and leukemic cells, making it an interesting target in AML. Here, we conducted a comprehensive immunomonitoring of a phase Ib trial (NCT03066648) evaluating decitabine in combination with anti-TIM3 antibody sabatolimab (MBG453). We studied paired bone marrow (BM) and peripheral blood samples from 11 unfit newly diagnosed (ND) or relapsed/refractory (R/R) AML patients and 1 MDS patient with single-cell RNA and T cell receptor sequencing (scRNA+TCRαβ-seq) and flow cytometry. We also performed co-culture assays of primary immune and leukemic cells with scRNA+TCRαβ-seq readout in a patient with a durable complete response. In scRNA+TCRαβ-seq data, HAVCR2 (encoding TIM3) is highly expressed in NK cells, myeloid cells, and unconventional T cells, with some expression in CD8+ T cells. This contrasts with PDCD1 (encoding PD1) and CTLA4, which are primarily expressed in CD8+ and CD4+ T cells, respectively, albeit at low levels in AML. At baseline, responders had higher proportions of CD4+ T cells and B lymphocytes, whereas non-responders had more cytotoxic CD8+ T cells, which was confirmed by flow cytometry. After starting anti-TIM3+HMA therapy, both groups showed increased numbers of CD8+ T cells and NK cells. Responders to anti-TIM3+HMA therapy generally had more mature (CD56dim and adaptive) NK cells, with key transcriptional changes in type I/II interferon (IFN) and NF-κB pathways. Functional co-culture assayswith primary blasts from a complete responder revealed three NK activation states: cell-contact activated, cytokine-secreting, and type I IFN-responsive. NK cells showed enhanced type I IFN responses after therapy correlating with clinical remission, but this response was reduced when NK cells were co-cultured with blasts from a relapse time point, suggesting potential immune evasion. Cytotoxic CD4+ T cells were more abundant and highly clonal in pre-treatment samples from responders. In an exceptional responder with concomitant CD4+ T-LGLL diagnosis, the dominant CD4+ T-LGLL clone comprised 21% of the TCR repertoire at baseline, persisted at remission, but fell to 7% at relapse. Transcriptomic analysis showed upregulation of IFNG and TNF post-therapy, with a subset peaking soon after treatment. To assess whether the T-LGLL clone targets patient's leukemic cells, we engineered Jurkat reporter cells with the TCRαβ from the CD4+ T-LGLL clone and performed co-culture assays with scRNA-seq readout. In comparison to mock transduced cells, T-LGLL reporter cells showed upregulation of type I IFN genes when co-cultured with patient's leukemia cells, especially in presence of antigen-presenting cells. In co-culture of patient's own T-LGLL cells, pre-therapy cells responded strongly to blasts from screening but lost reactivity to relapse blasts, while post-therapy T-LGLL cells retained activity to both. Exhausted CD8+ T cells were rare in AML BM (<0.5% pre-treatment), most CD8+ T cells exhibited effector or memory phenotypes. Functional co-culture assays showed that CD8+ T cells retained robust activation capacity, marked by TNF and IFNG expression, when co-cultured with autologous blasts. Anti-TIM3+HMA therapy preferentially expanded small CD8+ T cell clones in responders, whereas non-responders showed expansion of larger, more cytotoxic clones, some of which were recognized to target viral epitopes. ScRNA-seq analysis of myeloid cells revealed that responders had a higher proportion of differentiated myeloid cells, such as classical monocytes. In the exceptional responder, relapse blasts in co-culture upregulated immune evasion genes (e.g., CD274 encoding PDL1) and showed the highest HLA class I expression, indicating putative immune escape mechanisms from T and NK cells, respectively.Our study provides a comprehensive analysis of anti-TIM3 in combination with decitabine in AML/MDS. We demonstrate that TIM3 blockade modulates the immune landscape by activating mature and adaptive NK cells, promotes cytotoxic CD4+ T cells, and primes small CD8+ T cell clones for expansion. Our results suggest that cytotoxic CD4+ T-LGLL cells may boost responses to immune checkpoint therapy in AML.

The virtually monomorphic antigen presentation molecule HLA-E can present self- and non-self peptides to the NKG2A/CD94 co-receptor inhibitory complex expressed on natural killer (NK) cells and to T cell receptors (TCRs) expressed on T cells. HLA-E presents self-peptides to NKG2A/CD94 to regulate tissue homeostasis, whereas HLA-E restricted T cells mediate regulatory and cytotoxic responses toward pathogen-infected cells. In this study, we directly compared HLA-E/peptide recognition and signaling between NKG2A/CD94 and 2 HLA-E restricted TCRs that can recognize self-peptides or identical peptide mimics from the viral UL40 protein of cytomegalovirus using position substituted peptide variants. We show that position 7 is critical for interaction with NKG2A/CD94, whereas position 8 is important for interaction with the TCRs. The Arginine at position 5 of these peptides is an essential residue for recognition by both receptors. Thus, NKG2A/CD94 and TCRs have different requirements for recognition of peptides presented in HLA-E.

Monocytes and macrophages, as important constituents of the innate immune system, are equipped with multiple Toll-like-receptors (TLRs) to recognize invading pathogens, such as SARS-CoV-2, and mount an antiviral response. Nevertheless, their uncontrolled activation can lead to hyperinflammation seen in severe COVID-19. Surprisingly, we observed that recombinant SARS-CoV-2 Spike (S) and Nucleocapsid (N) proteins triggered only a weak proinflammatory response in human peripheral blood monocytes. By employing THP-1 and Jurkat NF-κB::eGFP reporter cell lines expressing specific TLRs, various TLR ligands and blocking antibodies, we determined that surface TLRs, including TLR2/1, TLR2/6 and TLR4 do not play a major role in SARS-CoV-2 sensing. However, monocytes are potently activated by the replication-competent SARS-CoV-2, and the response correlates with the viral uptake that is observed only in monocytes, but not in lymphocytes. We show that monocyte activation involves two distinct steps. Firstly, SARS-CoV-2 infects monocytes in a process independent of the S protein and the prime SARS-CoV-2 receptor angiotensin-converting enzyme 2. Instead, the alternative SARS-CoV-2 receptor CD147, which is highly expressed on monocytes, recognizes its well-known interaction partners cyclophilins A and B that are incorporated into SARS-CoV-2 virions. Secondly, upon viral uptake via the cyclophilin-CD147 interaction, that can be inhibited by specific CD147 blocking antibodies or competition with recombinant human cyclophilin A and B, SARS-CoV-2 RNA is recognized by TLR7/8 in endosomes, leading to upregulation of tumor necrosis factor (TNF), interleukin (IL)-1β and IL-6, comprising the core hyperinflammatory signature. Taken together, our data reveal a novel mechanism how human monocytes sense SARS-CoV-2 and suggest that targeting the cyclophilin-CD147 axis might be beneficial to alleviate overt myeloid-driven inflammation triggered by SARS-CoV-2 infection.

CD19 chimeric antigen receptor T (CD19CAR-T) cells have achieved promising outcomes in relapsed/refractory B cell malignancies. However, recurrences occur due to the loss of CAR-T cell persistence. We developed dual T/B cell co-stimulatory molecules (CD28 and CD40) in CAR-T cells to enhance intense tumoricidal activity and persistence. CD19.28.40z CAR-T cells promoted pNF-κB and pRelB downstream signaling while diminishing NFAT signaling upon antigen exposure. CD19.28.40z CAR-T cells demonstrated greater proliferation, which translated into effective anti-tumor cytotoxicity in long-term co-culture assay. Repetitive weekly antigen stimulation unveiled continuous CAR-T cell expansion while preserving central memory T cell subset and lower expression of exhaustion phenotypes. The intrinsic genes underlying CD19.28.40z CAR-T cell responses were compared with conventional CARs and demonstrated the up-regulated genes associated with T cell proliferation and memory as well as down-regulated genes related to apoptosis, exhaustion, and glycolysis pathway. Enrichment of genes toward T cell stemness, particularly SELL, IL-7r, TCF7, and KLF2, was observed. Effective and continuing anti-tumor cytotoxicity in vivo was exhibited in both B cell lymphoblastic leukemia and B cell non-Hodgkin lymphoma xenograft models while demonstrating persistent T cell memory signatures. The functional enhancement of CD37.28.40z CAR-T cell activities against CD37+ tumor cells was further validated. The modification of dual T/B cell signaling molecules remarkably maximized the efficacy of CAR-T cell therapy.

T cell co-inhibitory immune checkpoints, such as PD-1 or BTLA, are bona fide targets in cancer therapy. We used a human T cell reporter line to measure transcriptomic changes mediated by PD-1- and BTLA-induced signaling. T cell receptor (TCR)-complex stimulation resulted in the upregulation of a large number of genes but also in repression of a similar number of genes. PD-1 and BTLA signals attenuated transcriptomic changes mediated by TCR-complex signaling: upregulated genes tended to be suppressed and the expression of a significant number of downregulated genes was higher during PD-1 or BTLA signaling. BTLA was a significantly stronger attenuator of TCR-complex-induced transcriptome changes than PD-1. A strong overlap between genes that were regulated indicated quantitative rather than qualitative differences between these receptors. In line with their function as attenuators of TCR-complex-mediated changes, we found strongly regulated genes to be prime targets of PD-1 and BTLA signaling.

LAG-3 is a member of the immunoglobulin superfamily expressed on activated T cells, but also on other immune cells. It has significant homology to CD4. Both molecules have four extracellular Ig-like domains with similar structural motifs but the sequence identity between LAG-3 and CD4 is low. Furthermore, unlike CD4 LAG-3 restrains T cell responses and antibodies targeting this receptor are emerging drugs in cancer immunotherapy. A combination of LAG-3 and PD-1 antibodies has already been approved for the treatment of metastatic melanoma. Despite this success, its biology is still not well understood. Here we summarize the current knowledge on expression, ligands, and function of LAG-3. We point to the differences between LAG-3 and other inhibitory immune checkpoints and describe obstacles to study the role of this receptor in T cell activation processes. Finally, we discuss future directions for scientific efforts to come to a more complete understanding of the biology of this eminent immune checkpoint.

Understanding human T-cell antigen recognition in health and disease is becoming increasingly instrumental for monitoring T-cell responses to pathogen challenge and for the rational design of T-cell-based therapies targeting cancer, autoimmunity and organ transplant rejection. Here we showcase a quantitative imaging platform which is based on the use of planar glass-supported lipid bilayers (SLBs). The latter are functionalized with antigen (peptide-loaded HLA) as adhesion and costimulatory molecules (ICAM-1, B7-1) to serve as surrogate antigen presenting cell for antigen recognition by T-cells, which are equipped with T-cell antigen receptors (TCRs) sequenced from antigen-specific patient T-cells. We outline in detail, how the experimental use of SLBs supports recoding and analysis of synaptic antigen engagement and calcium signaling at the single cell level in response to user-defined antigen densities for quantitative comparison.

Antibodies can block immune receptor engagement or trigger the receptor machinery to initiate signaling. We hypothesized that antibody agonists trigger signaling by sterically excluding large receptor-type protein tyrosine phosphatases (RPTPs) such as CD45 from sites of receptor engagement. An agonist targeting the costimulatory receptor CD28 produced signals that depended on antibody immobilization and were sensitive to the sizes of the receptor, the RPTPs, and the antibody itself. Although both the agonist and a non-agonistic anti-CD28 antibody locally excluded CD45, the agonistic antibody was more effective. An anti-PD-1 antibody that bound membrane proximally excluded CD45, triggered Src homology 2 domain-containing phosphatase 2 recruitment, and suppressed systemic lupus erythematosus and delayed-type hypersensitivity in experimental models. Paradoxically, nivolumab and pembrolizumab, anti-PD-1-blocking antibodies used clinically, also excluded CD45 and were agonistic in certain settings. Reducing these agonistic effects using antibody engineering improved PD-1 blockade. These findings establish a framework for developing new and improved therapies for autoimmunity and cancer.

Background Many cancer immunotherapies rely on cell surface protein engagements in trans. Checkpoint inhibitors block certain interactions between cancer cells and immune cells. Chimeric antigen receptor (CAR)-based therapies redirect immune cells towards tumors by binding of surface antigens on cancer cells. This highlights the importance of such cellular interactions and provides a rationale to develop an easy-to-handle, flexible and cost-effective tool to study them. Here we present a novel immunoassay that fulfills these requirements and we show its utility to address research questions arising in the context of immunotherapies.1 Materials and Methods Our assay makes use of T cell lines equipped with reporter genes, that are translated upon activation of the TCR signaling pathway.2 To create cellular biosensors, the reporter cells are transduced to express chimeric receptors. These receptors consist of an extracellular domain of interest fused to a CD3ζ intracellular signaling domain. The cellular biosensors can then be probed with cells expressing the respective interacting receptor or ligand. This induces a fluorescent signal that is evaluable by flow cytometry. Results The assay provides quantitative information on the inhibitory potency of immune checkpoint inhibitors to the PD-L1/PD1 axis and can be used to characterize small molecule inhibitors blocking the PD1/PD-L1 interaction. While some are highly active, surprisingly others do not interfere with PD1 binding to PD-L1 even at high concentrations. We demonstrate that cellular biosensors can function to probe defined cellular populations for the expression of interaction partners to orphan ligands. In this setting, we do not identify a receptor to the putative immune checkpoint B7-H3 on T cells suggesting alternative mechanisms for B7-H3 mediated immunosuppression. Finally, we apply cellular biosensors to validate binding of CAR antigen recognition domain. Variable chains of three B7-H3 specific antibodies are arranged as single chain fragments (scFv) and evaluated in a cellular biosensor assay. While a mirzotamab derived scFv induces a strong signal, binding of an omburtabmab-scFv is considerably weaker and no signal can be detected with an enoblituzumab derived scFv. Conclusions In summary, we present a novel type of immunoassay to study interactions between membrane proteins in trans and highlight potential uses in the context of immunotherapy. It is suitable to study immune checkpoints and can be used to characterize respective inhibitory drugs. It also enables unbiased screening of cellular samples for the expression of interaction partners to orphan ligands. Lastly, it is applicable for the validation of antigen binding domains for CARs. This may accelerate the development of new, functional CAR constructs and could furthermore serve as a method to characterize the role of antigen mutations for immune escape. References Funk MA, Leitner J, Gerner MC, et al. Interrogating ligand-receptor interactions using highly sensitive cellular biosensors. Nat Commun. 2023;14:7804. Jutz S, Hennig A, Paster W, et al. A cellular platform for the evaluation of immune checkpoint molecules. Oncotarget. 2017;8(39):64892-64906. M.A. Funk: None. J. Leitner: None. C. Battin: None. S. Gumpelmair: None. S. Theurich: None. P. Steinberger: None.

Chimeric antigen receptors (CARs) equipped with an inhibitory signaling domain (iCARs) have been proposed as strategy to increase on-tumor specificity of CAR-T cell therapies. iCARs inhibit T cell activation upon antigen recognition and thereby program a Boolean NOT gate within the CAR-T cell. If cancer cells do not express the iCAR target antigen while it is highly expressed on healthy tissue, CAR/iCAR coexpressing T cells are supposed to kill cancer cells but not healthy cells expressing the CAR antigen. In this study, we employed a well-established reporter cell system to demonstrate high potency of iCAR constructs harboring BTLA-derived signaling domains. We then created CAR/iCAR combinations for the clinically relevant antigen pairs B7-H3/CD45 and CD123/CD19 and show potent reporter cell suppression by iCARs targeting CD45 or CD19. In primary human T cells αCD19-iCARs were capable of suppressing T cell proliferation and cytokine production. Surprisingly, the iCAR failed to veto immediate CAR-mediated cytotoxicity. Likewise, T cells overexpressing PD-1 or BTLA did not show impaired cytotoxicity toward ligand-expressing target cells, indicating that inhibitory signaling by these receptors does not mediate protection against cytotoxicity by CAR-T cells. Future approaches employing iCAR-equipped CAR-T cells for cancer therapy should therefore monitor off-tumor reactivity and potential CAR/iCAR-T cell dysfunction.

HLA-E molecules can present self- and pathogen-derived peptides to both natural killer (NK) cells and T cells. T cells that recognize HLA-E peptides via their T cell receptor (TCR) are termed donor-unrestricted T cells due to restricted allelic variation of HLA-E. The composition and repertoire of HLA-E TCRs is not known so far. We performed TCR sequencing on CD8+ T cells from 21 individuals recognizing HLA-E tetramers (TMs) folded with two Mtb-HLA-E-restricted peptides. We sorted HLA-E Mtb TM+ and TM- CD8+ T cells directly ex vivo and performed bulk RNA-sequencing and single-cell TCR sequencing. The identified TCR repertoire was diverse and showed no conservation between and within individuals. TCRs selected from our single-cell TCR sequencing data could be activated upon HLA-E/peptide stimulation, although not robust, reflecting potentially weak interactions between HLA-E peptide complexes and TCRs. Thus, HLA-E-Mtb-specific T cells have a highly diverse TCR repertoire.

Chimeric antigen receptor (CAR) T cell is a promising therapy for cancer, but factors that enhance the efficacy of CAR T cell remain elusive. Here we perform a genome-wide CRISPR screening to probe genes that regulate the proliferation and survival of CAR T cells following repetitive antigen stimulations. We find that genetic ablation of CUL5, encoding a core element of the multi-protein E3 ubiquitin-protein ligase complex, cullin-RING ligase 5, enhances human CD19 CAR T cell expansion potential and effector functions, potentially via the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. In this regard, CUL5 knockout CD19 CAR T cells show sustained STAT3 and STAT5 phosphorylation, as well as delayed phosphorylation and degradation of JAK1 and JAK3. In vivo, shRNA-mediated knockdown of CUL5 enhances CD19 CAR T treatment outcomes in tumor-bearing mice. Our findings thus imply that targeting CUL5 in the ubiquitin system may enhance CAR T cell effector functions to enhance immunotherapy efficacy.

Costimulatory signals provided to T cells during antigen encounter have a decisive role in the outcome of immune responses. Here, we used chimeric receptors harboring the extracellular domain of mouse inducible T cell costimulator (mICOS) to study transcriptional activation mediated by cytoplasmic sequences of the major T cell costimulatory receptors CD28, 4-1BB, and CD2. The chimeric receptors were introduced in a T cell reporter platform that allows to simultaneously evaluate nuclear factor κB (NF-κB), NFAT, and AP-1 activation. Engagement of the chimeric receptors induced distinct transcriptional profiles. CD28 signaling activated all three transcription factors, whereas 4-1BB strongly promoted NF-κB and AP-1 but downregulated NFAT activity. CD2 signals resulted in the strongest upregulation of NFAT. Transcriptome analysis revealed pronounced and distinct gene expression signatures upon CD2 and 4-1BB signaling. Using the intracellular sequence of CD28, we exemplify that distinct signaling motifs endow chimeric receptors with different costimulatory capacities.

The receptor for advanced glycation end products (RAGE) is encoded by AGER, a gene that is subjected to tissue-specific alternative splicing. Splice variants of RAGE in intestine and placenta are unknown and contradictory data concerning RAGE protein expression in these tissues have been published. As a basis for future functional studies, we examined RAGE expression in small intestine, colon and placentas. PCR cloning revealed that full-length RAGE is the only RAGE transcript isoform expressed in placenta. In the small intestine, the major transcript isoform detected was RAGE_v1 encoding the C-terminally truncated soluble receptor. In the colon, both full-length RAGE as well as several splice variants were identified. Four antibodies were used to study protein expression by immunoblotting and were carefully validated. Appropriate controls were essential to avoid misinterpretation of bands caused by non-specific reactivity of antibodies. Only one of four antibodies tested detected full-length RAGE in placenta, whereas no RAGE-specific band was detected in intestinal tissues despite loading >30-fold more intestinal tissue than the positive control, human lung. RAGE expression levels in the placenta were 100-fold lower compared with human lung when analyzed by ELISA, and no significant differences in RAGE expression were detected between healthy placentas and placentas from women with preeclampsia, gestational diabetes mellitus, or fetal growth restriction. We conclude that healthy placental chorionic tissue expresses low levels of full-length RAGE, whereas expression of the tissue-specific intestinal isoforms is below the limit of detection. Low RAGE expression levels in combination with a lack of antibody validation may explain the conflicting published results on RAGE protein expression in intestine and placenta.

Interactions of membrane-resident proteins are important targets for therapeutic interventions but most methods to study them are either costly, laborious or fail to reflect the physiologic interaction of membrane resident proteins in trans. Here we describe highly sensitive cellular biosensors as a tool to study receptor-ligand pairs. They consist of fluorescent reporter cells that express chimeric receptors harboring ectodomains of cell surface molecules and intracellular signaling domains. We show that a broad range of molecules can be integrated into this platform and we demonstrate its applicability to highly relevant research areas, including the characterization of immune checkpoints and the probing of cells for the presence of receptors or ligands. The platform is suitable to evaluate the interactions of viral proteins with host receptors and to test for neutralization capability of drugs or biological samples. Our results indicate that cellular biosensors have broad utility as a tool to study protein-interactions.

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.