References

The function of a T cell depends on its subtype and activation state. Here, we show that imaging of the autofluorescence lifetime signals of quiescent and activated T cells can be used to classify the cells. T cells isolated from human peripheral blood and activated in culture using tetrameric antibodies against the surface ligands CD2, CD3 and CD28 showed specific activation-state-dependent patterns of autofluorescence lifetime. Logistic regression models and random forest models classified T cells according to activation state with 97-99{\%} accuracy, and according to activation state (quiescent or activated) and subtype (CD3+CD8+ or CD3+CD4+) with 97{\%} accuracy. Autofluorescence lifetime imaging can be used to non-destructively determine T-cell function. View Publication

Pulmonary sarcoidosis has unknown etiology, a difficult diagnostic procedure and no curative treatment. Extracellular vesicles including exosomes are nano-sized entities released from all cell types. Previous studies of exosomes from bronchoalveolar lavage fluid (BALF) of sarcoidosis patients have revealed pro-inflammatory components and abilities, but cell sources and mechanisms have not been identified. In the current study, we found that BALF exosomes from sarcoidosis patients, but not from healthy individuals, induced a dose-dependent elevation of intracellular IL-1$\beta$ in monocytes. Analyses of supernatants showed that patient exosomes also induced release of IL-1$\beta$, IL-6 and TNF from both PBMCs and enriched monocytes, suggesting that the observed effect is direct on monocytes. The potently chemotactic chemokine CCL2 was induced by exosomes from a subgroup of patients, and in a blocking assay the exosome-induced CCL2 was reduced for 13 out of 19 patients by the asthma drug Montelukast, a cysteinyl leukotriene receptor antagonist. Further, reactive oxygen species generation by PBMCs was induced to a higher degree by patient exosomes compared to healthy exosomes. These findings add to an emerging picture of exosomes as mediators and disseminators of inflammation, and open for further investigations of the link between CCL2 and exosomal leukotrienes in sarcoidosis. View Publication

To avoid innate sensing and immune control, human immunodeficiency virus type 1 (HIV-1) has to prevent the accumulation of viral complementary DNA species. Here, we show that the late HIV-1 accessory protein Vpu hijacks DNA repair mechanisms to promote degradation of nuclear viral cDNA in cells that are already productively infected. Vpu achieves this by interacting with RanBP2-RanGAP1*SUMO1-Ubc9 SUMO E3-ligase complexes at the nuclear pore to reprogramme promyelocytic leukaemia protein nuclear bodies and reduce SUMOylation of Bloom syndrome protein, unleashing end degradation of viral cDNA. Concomitantly, Vpu inhibits RAD52-mediated homologous repair of viral cDNA, preventing the generation of dead-end circular forms of single copies of the long terminal repeat and permitting sustained nucleolytic attack. Our results identify Vpu as a key modulator of the DNA repair machinery. We show that Bloom syndrome protein eliminates nuclear HIV-1 cDNA and thereby suppresses immune sensing and proviral hyper-integration. Therapeutic targeting of DNA repair may facilitate the induction of antiviral immunity and suppress proviral integration replenishing latent HIV reservoirs. View Publication

A leading pharmacological strategy toward HIV cure requires shock" or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7) releasing active P-TEFb which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD) inducing HIV transcription." View Publication

Imaging strategies to monitor chimeric antigen receptor (CAR) T-cell biodistribution and proliferation harbor the potential to facilitate clinical translation for the treatment of both liquid and solid tumors. In addition, the potential adverse effects of CAR T cells highlight the need for mechanisms to modulate CAR T-cell activity. The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene has previously been translated as a PET reporter gene for imaging of T-cell trafficking in patients with brain tumor. The HSV1-TK enzyme can act as a suicide gene of transduced cells through treatment with the prodrug ganciclovir. Here we report the molecular engineering, imaging, and ganciclovir-mediated destruction of B7H3 CAR T cells incorporating a mutated version of the HSV1-tk gene (sr39tk) with improved enzymatic activity for ganciclovir. The sr39tk gene did not affect B7H3 CAR T-cell functionality and in vitro and in vivo studies in osteosarcoma models showed no significant effect on B7H3 CAR T-cell antitumor activity. PET/CT imaging with 9-(4-[18F]-fluoro-3-[hydroxymethyl]butyl)guanine ([18F]FHBG) of B7H3-sr39tk CAR T cells in an orthotopic model of osteosarcoma revealed tumor homing and systemic immune expansion. Bioluminescence and PET imaging of B7H3-sr39tk CAR T cells confirmed complete tumor ablation with intraperitoneal ganciclovir administration. This imaging and suicide ablation system can provide insight into CAR T-cell migration and proliferation during clinical trials while serving as a suicide switch to limit potential toxicities. SIGNIFICANCE: This study showcases the only genetically engineered system capable of serving the dual role both as an effective PET imaging reporter and as a suicide switch for CAR T cells. View Publication

During surgery, ATP from damaged cells induces the release of interleukin-1$\beta$, a potent pro-inflammatory cytokine that contributes to the development of postoperative systemic inflammation, sepsis and multi-organ damage. We recently demonstrated that C-reactive protein (CRP) inhibits the ATP-induced release of monocytic interleukin-1$\beta$, although high CRP levels are deemed to be a poor prognostic marker. Here, we retrospectively investigated if preoperative CRP levels correlate with postoperative CRP, leukocyte counts and fever in the context of anatomical lung resection and systematic lymph node dissection as first line lung cancer therapy. No correlation was found in the overall results. In men, however, preoperative CRP and leukocyte counts positively correlated on postoperative days one to two, and a negative correlation of CRP and fever was seen in women. These correlations were more pronounced in men taking statins and in statin-na{\{i}}ve women. Accordingly the inhibitory effect of CRP on the ATP-induced interleukin-1$\beta$ release was blunted in monocytes from coronary heart disease patients treated with atorvastatin compared to monocytes obtained before medication. Hence the common notion that elevated CRP levels predict more severe postoperative inflammation should be questioned. We rather hypothesize that in women and statin-na{\"{i}}ve patients high CRP levels attenuate trauma-induced increases in inflammatory markers.""" View Publication

A safe and effective vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be required to end the coronavirus disease 2019 (COVID-19) pandemic1-8. For global deployment and pandemic control, a vaccine that requires only a single immunization would be optimal. Here we show the immunogenicity and protective efficacy of a single dose of adenovirus serotype 26 (Ad26) vector-based vaccines expressing the SARS-CoV-2 spike (S) protein in non-human primates. Fifty-two rhesus macaques (Macaca mulatta) were immunized with Ad26 vectors that encoded S variants or sham control, and then challenged with SARS-CoV-2 by the intranasal and intratracheal routes9,10. The optimal Ad26 vaccine induced robust neutralizing antibody responses and provided complete or near-complete protection in bronchoalveolar lavage and nasal swabs after SARS-CoV-2 challenge. Titres of vaccine-elicited neutralizing antibodies correlated with protective efficacy, suggesting an immune correlate of protection. These data demonstrate robust single-shot vaccine protection against SARS-CoV-2 in non-human primates. The optimal Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in clinical trials. View Publication

As SARS-CoV-2 infections and death counts continue to rise, it remains unclear why some individuals recover from infection, whereas others rapidly progress and die. Although the immunological mechanisms that underlie different clinical trajectories remain poorly defined, pathogen-specific antibodies often point to immunological mechanisms of protection. Here, we profiled SARS-CoV-2-specific humoral responses in a cohort of 22 hospitalized individuals. Despite inter-individual heterogeneity, distinct antibody signatures resolved individuals with different outcomes. Although no differences in SARS-CoV-2-specific IgG levels were observed, spike-specific humoral responses were enriched among convalescent individuals, whereas functional antibody responses to the nucleocapsid were elevated in deceased individuals. Furthermore, this enriched immunodominant spike-specific antibody profile in convalescents was confirmed in a larger validation cohort. These results demonstrate that early antigen-specific and qualitative features of SARS-CoV-2-specific antibodies point to differences in disease trajectory, highlighting the potential importance of functional antigen-specific humoral immunity to guide patient care and vaccine development. View Publication

Several HIV-1 and SIV vaccine candidates have shown partial protection against viral challenges in rhesus macaques. However, the protective efficacy of vaccine-elicited polyclonal antibodies has not previously been demonstrated in adoptive transfer studies in nonhuman primates. In this study, we show that passive transfer of purified antibodies from vaccinated macaques can protect naive animals against SIVmac251 challenges. We vaccinated 30 rhesus macaques with Ad26-SIV Env/Gag/Pol and SIV Env gp140 protein vaccines and assessed the induction of antibody responses and a putative protective signature. This signature included multiple antibody functions and correlated with upregulation of interferon pathways in vaccinated animals. Adoptive transfer of purified immunoglobulin G (IgG) from the vaccinated animals with the most robust protective signatures provided partial protection against SIVmac251 challenges in naive recipient rhesus macaques. These data demonstrate the protective efficacy of purified vaccine-elicited antiviral antibodies in this model, even in the absence of virus neutralization. View Publication

The ability to analyze protein function in a native context is central to understanding cellular physiology. This study explores whether tagging endogenous proteins with a reporter is a scalable strategy for generating cell models that accurately quantitate protein dynamics. Specifically, it investigates whether CRISPR-mediated integration of the HiBiT luminescent peptide tag can easily be accomplished on a large-scale and whether integrated reporter faithfully represents target biology. For this purpose, a large set of proteins representing diverse structures and functions, some of which are known or potential drug targets, were targeted for tagging with HiBiT in multiple cell lines. Successful insertion was detected for 86{\%} of the targets, as determined by luminescence-based plate assays, blotting, and imaging. In order to determine whether endogenously tagged proteins yield more representative models, cells expressing HiBiT protein fusions either from endogenous loci or plasmids were directly compared in functional assays. In the tested cases, only the edited lines were capable of accurately reproducing the anticipated biology. This study provides evidence that cell lines expressing HiBiT fusions from endogenous loci can be rapidly generated for many different proteins and that these cellular models provide insight into protein function that may be unobtainable using overexpression-based approaches. View Publication

Hepatocellular carcinoma (HCC) is among the most common causes of cancer death in men. Whether or not a longitudinal follow-up of circulating tumor cells (CTCs) before and at different time points during systemic/targeted therapy is useful for monitoring the treatment response of patients with locally advanced or metastatic HCC has been evaluated in this study. Blood samples (n = 104) were obtained from patients with locally advanced or metastatic HCC (n = 30) for the enrichment of CTCs by a negative selection method. Analysis of the blood samples from patients with defined disease status (n = 81) revealed that those with progressive disease (PD, n = 37) had significantly higher CTC counts compared to those with a partial response (PR) or stable disease (SD; n = 44 for PR + SD, p = 0.0002). The median CTC count for patients with PD and for patients with PR and SD was 50 (interquartile range 21-139) and 15 (interquartile range 4-41) cells/mL of blood, respectively. A longitudinal analysis of patients (n = 17) after a series of blood collections demonstrated that a change in the CTC count correlated with the patient treatment response in most of the cases and was particularly useful for monitoring patients without elevated serum alpha-fetoprotein (AFP) levels. Sequential CTC enumeration during treatment can supplement standard medical tests and benefit the management of patients with locally advanced or metastatic HCC, in particular for the AFP-low cases. View Publication

Immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide show remarkable antitumor activity in multiple myeloma (MM) via directly inhibiting MM-cell growth in the bone marrow (BM) microenvironment and promoting immune effector cell function. They are known to bind to the ubiquitin 3 ligase CRBN complex and thereby triggering degradation of IKZF1/3. In this study, we demonstrate that IMiDs also directly bind and activate zeta-chain-associated protein kinase-70 (Zap-70) via its tyrosine residue phosphorylation in T cells. IMiDs also triggered phosphorylation of Zap-70 in natural killer (NK) cells. Importantly, increased granzyme-B (GZM-B) expression and NK-cell activity triggered by IMiDs is associated with Zap-70 activation and inhibited by Zap-70 knockdown (KD), independent of CRBN. We also demonstrate a second mechanism whereby IMiDs trigger GZM-B and NK cytotoxicity which is CRBN and IKZF3 mediated, and inhibited or enhanced by KD of CRBN or IKZF3, respectively, independent of Zap-70. Our studies therefore show that IMiDs can enhance NK and T-cell cytotoxicity in (1) ZAP-70-mediated CRBN independent, as well as (2) CRBN-mediated ZAP-70 independent mechanisms; and provide the framework for developing novel therapeutics to activate Zap-70 and thereby enhance T and NK anti-MM cytotoxicity. View Publication