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Ligand inducible proteins that enable precise and reversible control of nuclear translocation of passenger proteins have broad applications ranging from genetic studies in mammals to therapeutics that target diseases such as cancer and diabetes. One of the drawbacks of the current translocation systems is that the ligands used to control nuclear localization are either toxic or prone to crosstalk with endogenous protein cascades within live animals. We sought to take advantage of salicylic acid (SA), a small molecule that has been extensively used in humans. In plants, SA functions as a hormone that can mediate immunity and is sensed by the non-expressor of pathogenesis-related (NPR) proteins. Although it is well recognized that nuclear translocation of NPR1 is essential to promoting immunity in plants, the exact subdomain of Arabidopsis thaliana NPR1 (AtNPR1) essential for SA mediated nuclear translocation is controversial. Here, we utilized the fluorescent protein mCherry as the reporter to investigate the ability of SA to induce nuclear translocation of the full-length NPR1 protein or its C-terminal transactivation (TAD) domain using HEK293 cells as a heterologous system. HEK293 cells lack accessory plant proteins including NPR3/NPR4 and are thus ideally suited for studying the impact of SA-induced changes in NPR1. Our results obtained using a stable expression system show that the TAD of AtNPR1 is sufficient to enable the reversible SA mediated nuclear translocation of mCherry. Our studies advance a basic understanding of nuclear translocation mediated by the TAD of AtNPR1 and uncover a biotechnological tool for SA mediated nuclear localization. This article is protected by copyright. All rights reserved.

Engineering cellular therapeutics by programming T cells has great potential in immunology. The primary mechanism employed by T cells for the specific transfer of proteins at the immunological synapse is via the lysosomal perforin pathway that facilitates the transfer of cytotoxic granzymes leading to apoptosis in target cells. Facilitating the delivery of non-cytotoxic proteins through perforin oligomers will dramatically expand the range of protein cargos that T cells can traffic to the target cells. Here, we have identified the intralysosomal protein, NPC2, as a chaperone that can facilitate the delivery of T-cell derived reporter proteins through perforin pores at the immunological synapse. Structural and biophysical considerations suggested that NPC2 could traverse through perforin pores and in vitro experiments confirmed the transport of purified NPC2 through perforin pores on cell membranes. To characterize the ability of NPC2 to facilitate the transfer of payloads in T cells, we constructed NPC2-mCherry fusion proteins in T cells. Using confocal microscopy and flow cytometry, we confirmed the colocalization of the NPC2 fused protein with lytic granules and the transfer of the fluorescent protein payload from T cells to target cells in co-culture experiments. The NPC2 fusion enabled the localization of mCherry to secretory lysosomes in mouse TCR CD8+ T cells and human CD4+ and CD8+ chimeric antigen receptor (CAR) T cells. These results illustrate that by using NPC2 as a molecular chaperone, the NPC2-perforin pathway can be exploited as a programmable molecular delivery system for cell-based therapies.

Adoptive immunotherapy with T cells expressing chimeric antigen receptors (CARs) for B-cell malignancies serves as a model for identifying subsets with superior clinical activity. We profiled the infusion products (IP) of 16 patients with large B-cell lymphoma (LBCL) using an integrated suite of single-cell assays to reveal the therapeutic potential of CD19-specific CAR+ T cells. Timelapse imaging microscopy in nanowell grids (TIMING) profiling revealed that T cells from responders showed migration (persistent motion for at least one body length), and migration was associated with serial killing capacity. In addition, confocal microscopy revealed that migration is linearly correlated with both mitochondrial volume and lysosomal volume; and scRNA-seq demonstrated that T cells from responders were enriched in pathways related to T-cell killing, migration and actin cytoskeleton, and TCR clustering. A marker-free sorting strategy enriched T cells with migratory capacity and validated serial killing, bioenergetics, and in vivo efficacy. In aggregate, we demonstrate that migration is a cell-intrinsic biomarker independent of CAR design or biomanufacturing, desired in the bioactivity of CAR+ T cells associated with clinical antitumor efficacy.

The in vivo persistence of adoptively transferred T cells is predictive of anti-tumor response. Identifying functional properties of infused T cells that lead to in vivo persistence and tumor eradication has remained elusive. We profiled CD19-specific CAR T cells that comprise the infusion products used to treat large B cell lymphomas using high-throughput single-cell technologies based on Timelapse Imaging Microscopy In Nanowell Grids (TIMING) that integrates killing, cytokine secretion, and transcriptional profiling. Our results show that the directional migration of CD19-specific CAR T cells is correlated with polyfunctionality. We identified that CD2 on T cells is associated with directional migration and that the interaction between CD2 on T cells and CD58 on lymphoma cells accelerates killing and serial killing. Consistent with this, we observed elevated CD58 expression on pre-treatment tumor samples in patients with relapsed or refractory large B cell lymphomas treated with CD19-specific CAR T cell therapy was associated with complete clinical response and survival. These results highlight the importance of studying dynamic T-cell tumor cell interactions in identifying optimal antitumor responses.KEY POINTS- Profiling patient infusion products revealed that polyfunctional CAR T cells show directional migration, which is associated with higher CD2 expression- The ligand for CD2, CD58 is expressed at higher levels in the tumors of lymphoma patients who respond better to CAR T cell treatment

BackgroundAdoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive.MethodsTo identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling.ResultsWith the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107apos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice.ConclusionsBroadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit.

Background aims The genus Cryptococcus comprises two major fungal species that cause clinical infections in humans: Cryptococcus gattii and Cryptococcus neoformans. To establish invasive human disease, inhaled cryptococci must penetrate the lung tissue and reproduce. Each year, about 1 million cases of Cryptococcus infection are reported worldwide, and the infection's mortality rate ranges from 20% to 70%. Many HIV+/AIDS patients are affected by Cryptococcus infections, with 220,000 cases of cryptococcal meningitis reported worldwide in this population every year (C. neoformans infection statistics, via the Centers for Disease Control and Prevention, https://www.cdc.gov/fungal/diseases/cryptococcosis-neoformans/statistics.html ). To escape from host immune cell attack, Cryptococcus covers itself in a sugar-based capsule composed primarily of glucuronoxylomannan (GXM). To evade phagocytosis, yeast cells increase to a >45-µm perimeter and become titan, or giant, cells. Cryptococci virulence is directly proportional to the percentage of titan/giant cells present during Cryptococcus infection. To combat cryptococcosis, the authors propose the redirection of CD8+ T cells to target the GXM in the capsule via expression of a GXM-specific chimeric antigen receptor (GXMR-CAR). Results GXMR-CAR has an anti-GXM single-chain variable fragment followed by an IgG4 stalk in the extracellular domain, a CD28 transmembrane domain and CD28 and CD3-ς signaling domains. After lentiviral transduction of human T cells with the GXMR-CAR construct, flow cytometry demonstrated that 82.4% of the cells expressed GXMR-CAR on their surface. To determine whether the GXMR-CAR+ T cells exhibited GXM-specific recognition, these cells were incubated with GXM for 24 h and examined with the use of brightfield microscopy. Large clusters of proliferating GXMR-CAR+ T cells were observed in GXM-treated cells, whereas no clusters were observed in control cells. Moreover, the interaction of GXM with GXMR-CAR+ T cells was detected via flow cytometry by using a GXM-specific antibody, and the recognition of GXM by GXMR-CAR T cells triggered the secretion of granzyme and interferon gamma (IFN-γ). The ability of GXMR-CAR T cells to bind to the yeast form of C. neoformans was detected by fluorescent microscopy, but no binding was detected in mock-transduced control T cells (NoDNA T cells). Moreover, lung tissue sections were stained with Gomori Methenamine Silver and evaluated by NanoZoomer (Hamamatsu), revealing a significantly lower number of titan cells, with perimeters ranging from 50 to 130 µm and giant cells >130 µm in the CAR T-cell treated group when compared with other groups. Therefore, the authors validated the study's hypothesis by the redirection of GXMR-CAR+ T cells to target GXM, which induces the secretion of cytotoxic granules and IFN-γ that will aid in the control of cryptococcosis Conclusions Thus, these findings reveal that GXMR-CAR+ T cells can target C. neoformans. Future studies will be focused on determining the therapeutic efficacy of GXMR-CAR+ T cells in an animal model of cryptococcosis.

T-cell therapy with specificity redirected through chimeric antigen receptors (CARs) has shown efficacy for the treatment of hematologic malignancies. Although treatment with CAR T cell can result in high response rates, the properties of the cells that comprise the cellular infusion product, associated with clinical benefit are incompletely understood. We utilized a suite of high-throughput single-cell assays including single-cell RNA-sequencing (scRNA-seq); confocal microscopy; and Timelapse Imaging Microscopy In Nanowell Grids (TIMING). TIMING profiling of a cohort of 16 patients showed that persistent motility of T cells in the presence of tumor cells was associated with both serial killing capacity and polyfunctionality. Confocal microscopy on these same T cells revealed that persistent motility is linearly correlated with both mitochondrial volume and lysosomal number. ScRNA-seq demonstrated that T cells from responders were enriched in pathways related to T-cell proliferative capacity; interferon responses; and a distinct cluster of pathways related to actin cytoskeleton and migration. We employed a marker-free sorting strategy for enriching T cells with persistent motility. RNA-seq on sorted motile T cells showed an enrichment of the core motility signature. These motile T cells also demonstrated superior in vivo anti-leukemia efficacy in comparison to unsorted T cells. Lastly, we also confirmed the association with increased persistent motility and killing of CAR T cells across diverse CARs. In aggregate, our data identified that independent of CAR design or biomanufacturing, persistent motility serves as a selectable cell-intrinsic biomarker, desired in the bioactivity of expanded CAR+ T cells.

The genusCryptococcuscomprises two major fungal species that cause clinical infections in humans:C. gattiiandC. neoformans. To establish invasive human disease, inhaled Cryptococci must penetrate the lung tissue and reproduce. Each year, about 1 million cases ofCryptococcusinfection are reported worldwide, and the infection’s mortality rate ranges from 20% to 70%. HIV+/AIDS patients are highly susceptible toCryptococcusinfection. Therefore, we hypothesized that CD8+T cells could be redirected to target glucuronoxylomannan (GXM), a sugar present in theCryptococcusspecies capsule, via expression of a GXM-specific chimeric antigen receptor (GXMR-CAR) for treatment of cryptococcosis. GXMR-CAR has an anti-GXM single-chain variable fragment followed by an IgG4 stalk, a CD28 transmembrane domain, and CD3-ς and CD28 signaling domains. After lentiviral transduction of human T cells with the GXMR-CAR construct, flow cytometry demonstrated that 82.4% of the cells expressed GXMR-CAR on their surface. To determine whether the GXMR-CAR+T cells exhibited GXM-specific recognition, these cells were incubated with GXM for 24 h and examined using bright-field microscopy. Large clusters of proliferating GXMR-CAR+T cells were observed, while no clusters were present in the control cells. Moreover, the interaction of GXM with GXMR-CAR+T cells was detected via flow cytometry using a GXM-specific antibody. The ability of GXMR-CAR T cells to bind to the yeast form ofC. neoformanswas detected by fluorescent microscopy, but no binding was detected with NoDNA T cells. Furthermore, when GXMR-CAR+T cells were administered to immunocompromised NSG mice infected withC. neoformanstheirC. neoformansburden was significantly lower than mock-transduced control T cell treated mice as shown via immunofluorescence using an anti-GXM antibody and Gomori methenamine-silver (GMS) staining of Titan cells in lung tissue. Thus, these findings demonstrated the effectiveness of GXMR-CAR+T-cell therapy for cryptococcosis in a murine model.Author summaryCryptococcus gattiiinfects both immunocompetent and immunodeficient patients such as those with HIV/AIDS, whileC. neoformansusually infects only immunocompromised patients. Every year, almost one million HIV/AIDS patients suffer fromCryptococcusfungal co-infection. At present, no curative treatment is available to treat cryptococcosis in chronic HIV/AIDS patients. The objective of this research was to develop novel “Bioengineered”Cryptococcusspecific chimeric antigen receptor (CAR) CD8+T-cells to target and killCryptococcus. By using a culture model, we demonstrated that theCryptococcusspecific CAR T cells were able to bind to the yeast form ofC. neoformans.Using a mouse model ofCryptococcus, theCryptococcusspecific CAR treated group showed a significant reduction of fungal burden in lung tissue when compared to the control group. This gives new hope to HIV/AIDS patients suffering from cryptococcal infections.

Motivation Automated profiling of cell–cell interactions from high-throughput time-lapse imaging microscopy data of cells in nanowell grids (TIMING) has led to fundamental insights into cell–cell interactions in immunotherapy. This application note aims to enable widespread adoption of TIMING by (i) enabling the computations to occur on a desktop computer with a graphical processing unit instead of a server; (ii) enabling image acquisition and analysis to occur in the laboratory avoiding network data transfers to/from a server and (iii) providing a comprehensive graphical user interface. Results On a desktop computer, TIMING 2.0 takes 5 s/block/image frame, four times faster than our previous method on the same computer, and twice as fast as our previous method (TIMING) running on a Dell PowerEdge server. The cell segmentation accuracy (f-number = 0.993) is superior to our previous method (f-number = 0.821). A graphical user interface provides the ability to inspect the video analysis results, make corrective edits efficiently (one-click editing of an entire nanowell video sequence in 5–10 s) and display a summary of the cell killing efficacy measurements. Availability and implementation Open source Python software (GPL v3 license), instruction manual, sample data and sample results are included with the Supplement (https://github.com/RoysamLab/TIMING2). Supplementary information Supplementary data are available at Bioinformatics online.

T cells engineered to express chimeric antigen receptor (CAR) targeting CD19 have shown promising clinical responses in patients with certain hematologic malignancies, however, it is desirable to be able to enrich cells with enhanced anti-tumor efficacy prior to infusion. We utilized a suite of high-throughput technologies with single-cell resolution, including Timelapse Imaging Microscopy In Nanowell Grids (TIMING) that integrates cytokine profiling to reveal that persistent motility of CD19- specific CAR T cells is correlated to desirable polyfunctionality (elimination of tumor cells and cytokine secretion), contributing to anti-tumor effects. We implemented a marker-free Boyden chamber-based method to enrich CAR+ T cells with persistent motility (motile cell). Integration of transcriptomic profiling, immune phenotyping and metabolism demonstrated that motile cells are more naïve-like with higher oxidative metabolism and spare respiratory capacity. Our result also revealed that the master metabolic regulator AMP kinase (AMPK) is required for CAR+ T cells with high motility. We used a xenograft leukemia mouse model (CD19+ NALM-6) and validated that the motile cells have enhanced persistence and superior anti-cancer effect in vivo compared to the parental un-sorted population. Collectively, our multi-dimensional results demonstrated that persistent motility is a selectable biomarker of expanded CAR+ T cell bioactivity.

T cell-based therapies have shown promising results, but adoptive T cell therapy (ACT) for solid tumors like melanoma treatment has met limited success. The goal of this study was to identify attributes of T cell fitness important for optimal anti-tumor efficacy. This was accomplished by integrated profiling of expanded tumor infiltrating lymphocytes, TILs (responders (CR) and progressive diseases (PD)) co-cultured overnight with their autologous primary tumor cells with the aid of a suite of single-cell, transcriptional, proteomic and functional assays to construct a genome-scale metabolic model of the metabolism of TILs and tumor cells in direct competition with each other. Sorted live TILs after co-culture were used to performed RNA-seq, and proteome-wide profiling by mass spectrometry. Gene enrichment of the CR TIL suggested an increased expression in genes involving processes such as glycolysis, hypoxia, adipogenesis, and mTORC1 signaling. PD tumors showed an increase for epithelial to mesenchymal transition (EMT) and glycolysis. Comparisons of transcripts and proteins in both CR and PD were tightly correlated (Spearman rank 0.765). Since patient-derived cell numbers were limited (

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults with a dismal prognosis. Current therapy of surgical removal combined with Temozolomide (TMZ) and radiation therapy only slightly prolongs the survival of GBM patients. Thus, it is essential to elucidate mechanism underlying its highly malignant properties in order to develop efficacious therapeutic regimens. In this study, we showed that progranulin (PGRN) was overexpressed in most GBM cell lines and the majority of human tumor samples. PGRN overexpression conferred GBM cells with tumorigenic properties and TMZ resistance by upregulating DNA repair (PARP, ATM, BRCA1, Rad51, XRCC1 and so on) and cancer stemness (CD133, CD44, ABCG2) genes, in part via an AP-1 transcription factor, specifically cFos/JunB. Curcumin, an AP-1 inhibitor, was also found to regulate PGRN promoter activity and expression including its downstream effectors aforementioned. These data suggested a feedforward loop between PGRN signaling and AP-1. PGRN depletion significantly decreased unlimited self-renewal and multilineage differentiation and the malignant properties of GBMs cells S1R1, and enhanced their vulnerability to TMZ. In addition, S1R1 depleted of PGRN also lost the ability to form tumor in an orthotopic xenograft mouse model. In conclusion, PGRN had a critical role in the pathogenesis and chemoresistance of GBM and functioned at the top of the hierarchy of cellular machinery that modulates both DNA repair pathways and cancer stemness. Our data suggest that a new strategy combining current regimens with compounds targeting PGRN/AP-1 loop like curcumin may significantly improve the therapeutic outcome of GBM.

The transcription termination factor Rho of Escherichia coli is a RNA binding protein which can translocate along the RNA and unwind the RNA:DNA hybrid using the RNA-dependent ATPase activity. In order to investigate the involvement of each of these functions in releasing RNA from the elongation complex, we have isolated different termination defective mutants of Rho by random mutagenesis, characterized them for their different functions and established the structure–function correlations from the available structural data of Rho. These mutations are located within the two domains; the N-terminal RNA binding domain (G51V, G53V, and Y80C) and in the C-terminal ATP binding domain (Y274D, P279S, P279L, G324D, N340S, I382N) including the two important structural elements, the Q-loop (P279S, P279L) and R-loop (G324D). Termination defects of the mutants in primary RNA binding domain and Q-loop could not be restored under any conditions that we tested and these were also defective for most of the other functions of Rho. The termination defects of the mutants (Y274D, G324D and N340S), which were mainly defective for secondary RNA binding and likely defective for translocase activity, could be restored under relaxed in vitro conditions. We also show that a mutation in a primary RNA binding domain (Y80C) can cause a defect in ATP binding and induce distinct conformational changes in the distal C-terminal domain, and these allosteric effects are not predictable from the crystal structure. We conclude that the interactions in the primary RNA binding domain and in the Q-loop are mandatory for RNA release to occur and propose that the interactions in the primary RNA binding modulate most of the other functions of Rho allosterically. The rate of ATP hydrolysis regulates the processivity of translocation along the RNA and is directly correlated with the efficiency of RNA release. NusG improves the speed of RNA release and is not involved in any other step.