The Association of Low CD4 Expression on Monocytes and Low CD8+ T-Cell Count at Hospital Admission Predicts the Need for Mechanical Ventilation in Patients With COVID-19 Pneumonia: A Prospective Monocentric Cohort Study.

To identify COVID-19-associated immunophenotyping patterns at hospital admission and to determine if some patterns could predict the need for mechanical ventilation (MV). DESIGN: Prospective observational monocentric cohort study. SETTING: A university-affiliated hospital in Marseille, France. PATIENTS: Thirty patients presenting with laboratory-confirmed COVID-19 pneumonia were enrolled within the first 48 hours of hospital admission and compared with 18 healthy controls. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Whole-blood leukocytes were immunophenotyped with a rapid and simplified one-step flow cytometry method. Thirty-eight immune and five laboratory parameters were compared first between COVID-19 patients and controls and then between the COVID-19 patients who received or not MV during their stays. The variables that significantly discriminated MV from non-MV patients in univariate analysis were entered into a multiple stepwise logistic regression analysis. The COVID-19 patients were predominantly male (87%), aged 61 years (50-71 yr), and 93% received early corticosteroid therapy. Sixteen patients (53%) were managed with noninvasive respiratory support, and 14 (47%) required MV. Compared with controls, COVID-19 patients were characterized by an immune signature featuring: 1) decreased HLA-DR expression on monocytes; 2) reduced basophils, eosinophils, T-cells, NK cells, and nonclassical monocyte count; and 3) up regulation of CD169 on monocytes, CD64 on neutrophils, the adhesion/migration markers (CD62L and CD11b), and the checkpoint inhibitor CD274 on myeloid cells. Among the COVID-19 patients, those who received MV had lower level of CD4 and HLA-DR on monocytes, lower CD8+ T-cell count, and higher lactate dehydrogenase at hospital admission. In multivariate analysis, only CD4 on monocytes (p = 0.032) and CD8+ T-cell count (p = 0.026) were associated with MV requirement. The model combining these two variables provided an area under curve of 0.97 (95% CI, 0.83-0.99). CONCLUSIONS: The association of low CD4 on monocytes and low CD8+ T-cell count at hospital admission was highly predictive of the need for MV in hospitalized patients with COVID-19 pneumonia.

A rapid, easy, and scalable whole blood monocyte CD169 assay for outpatient screening during SARS-CoV-2 outbreak, and potentially other emerging disease outbreaks.

Objective: The COVID-19 corona virus disease outbreak is globally challenging health systems and societies. Its diagnosis relies on molecular methods, with drawbacks revealed by mass screening. Upregulation of neutrophil CD64 or monocyte CD169 has been abundantly reported as markers of bacterial or acute viral infection, respectively. We evaluated the sensitivity of an easy, one-step whole blood flow cytometry assay to measure these markers within 10 min, as a potential screening test for COVID-19 patients. Methods: Patients (n = 177) with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were tested on 10 µL blood and results were compared with reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). Results: We observed 98% and 100% sensitivity in early-stage (n = 52) and asymptomatic patients (n = 9), respectively. Late-stage patients, who presented for a second control RT-qPCR, were negative for both assays in most cases. Conversely, neutrophil CD64 expression was unchanged in 75% of cases, without significant differences between groups. Conclusion: Monocyte CD169 evaluation was highly sensitive for detecting SARS-CoV-2 infection in first-presentation patients; and it returns to basal level upon infection clearance. The potential ease of fingerprick collection, minimal time-to-result, and low cost rank this biomarker measurement as a potential viral disease screening tool, including COVID-19. When the virus prevalence in the tested population is usually low (1%-10%), such an approach could increase the testing capacity 10 to 100-fold, with the same limited molecular testing resources, which could focus on confirmation purposes only.

Advanced Flow Cytometry Assays for Immune Monitoring of CAR-T Cell Applications.

Adoptive immunotherapy using chimeric antigen receptor (CAR)-T cells has achieved successful remissions in refractory B-cell leukemia and B-cell lymphomas. In order to estimate both success and severe side effects of CAR-T cell therapies, longitudinal monitoring of the patient's immune system including CAR-T cells is desirable to accompany clinical staging. To conduct research on the fate and immunological impact of infused CAR-T cells, we established standardized 13-colour/15-parameter flow cytometry assays that are suitable to characterize immune cell subpopulations in the peripheral blood during CAR-T cell treatment. The respective staining technology is based on pre-formulated dry antibody panels in a uniform format. Additionally, further antibodies of choice can be added to address specific clinical or research questions. We designed panels for the anti-CD19 CAR-T therapy and, as a proof of concept, we assessed a healthy individual and three B-cell lymphoma patients treated with anti-CD19 CAR-T cells. We analyzed the presence of anti-CD19 CAR-T cells as well as residual CD19+ B cells, the activation status of the T-cell compartment, the expression of co-stimulatory signaling molecules and cytotoxic agents such as perforin and granzyme B. In summary, this work introduces standardized and modular flow cytometry assays for CAR-T cell clinical research, which could also be adapted in the future as quality controls during the CAR-T cell manufacturing process.

A Granulocytic Signature Identifies COVID-19 and Its Severity.

BACKGROUND: An unbiased approach to SARS-CoV-2-induced immune dysregulation has not been undertaken so far. We aimed to identify previously unreported immune markers able to discriminate COVID-19 patients from healthy controls and to predict mild and severe disease. METHODS: An observational, prospective, multicentric study was conducted in patients with confirmed mild/moderate (n = 7) and severe (n = 19) COVID-19. Immunophenotyping of whole-blood leukocytes was performed in patients upon hospital ward or intensive care unit admission and in healthy controls (n = 25). Clinically relevant associations were identified through unsupervised analysis. RESULTS: Granulocytic (neutrophil, eosinophil, and basophil) markers were enriched during COVID-19 and discriminated between patients with mild and severe disease. Increased counts of CD15+CD16+ neutrophils, decreased granulocytic expression of integrin CD11b, and Th2-related CRTH2 downregulation in eosinophils and basophils established a COVID-19 signature. Severity was associated with emergence of PD-L1 checkpoint expression in basophils and eosinophils. This granulocytic signature was accompanied by monocyte and lymphocyte immunoparalysis. Correlation with validated clinical scores supported pathophysiological relevance. CONCLUSIONS: Phenotypic markers of circulating granulocytes are strong discriminators between infected and uninfected individuals as well as between severity stages. COVID-19 alters the frequency and functional phenotypes of granulocyte subsets with emergence of CRTH2 as a disease biomarker.

Regulatory cell therapy in kidney transplantation (The ONE Study): a harmonised design and analysis of seven non-randomised, single-arm, phase 1/2A trials.

BACKGROUND: Use of cell-based medicinal products (CBMPs) represents a state-of-the-art approach for reducing general immunosuppression in organ transplantation. We tested multiple regulatory CBMPs in kidney transplant trials to establish the safety of regulatory CBMPs when combined with reduced immunosuppressive treatment. METHODS: The ONE Study consisted of seven investigator-led, single-arm trials done internationally at eight hospitals in France, Germany, Italy, the UK, and the USA (60 week follow-up). Included patients were living-donor kidney transplant recipients aged 18 years and older. The reference group trial (RGT) was a standard-of-care group given basiliximab, tapered steroids, mycophenolate mofetil, and tacrolimus. Six non-randomised phase 1/2A cell therapy group (CTG) trials were pooled and analysed, in which patients received one of six CBMPs containing regulatory T cells, dendritic cells, or macrophages; patient selection and immunosuppression mirrored the RGT, except basiliximab induction was substituted with CBMPs and mycophenolate mofetil tapering was allowed. None of the trials were randomised and none of the individuals involved were masked. The primary endpoint was biopsy-confirmed acute rejection (BCAR) within 60 weeks after transplantation; adverse event coding was centralised. The RTG and CTG trials are registered with ClinicalTrials.gov, NCT01656135, NCT02252055, NCT02085629, NCT02244801, NCT02371434, NCT02129881, and NCT02091232. FINDINGS: The seven trials took place between Dec 11, 2012, and Nov 14, 2018. Of 782 patients assessed for eligibility, 130 (17%) patients were enrolled and 104 were treated and included in the analysis. The 66 patients who were treated in the RGT were 73% male and had a median age of 47 years. The 38 patients who were treated across six CTG trials were 71% male and had a median age of 45 years. Standard-of-care immunosuppression in the recipients in the RGT resulted in a 12% BCAR rate (expected range 3·2-18·0). The overall BCAR rate for the six parallel CTG trials was 16%. 15 (40%) patients given CBMPs were successfully weaned from mycophenolate mofetil and maintained on tacrolimus monotherapy. Combined adverse event data and BCAR episodes from all six CTG trials revealed no safety concerns when compared with the RGT. Fewer episodes of infections were registered in CTG trials versus the RGT. INTERPRETATION: Regulatory cell therapy is achievable and safe in living-donor kidney transplant recipients, and is associated with fewer infectious complications, but similar rejection rates in the first year. Therefore, immune cell therapy is a potentially useful therapeutic approach in recipients of kidney transplant to minimise the burden of general immunosuppression. FUNDING: The 7th EU Framework Programme.

Standardization of whole blood immune phenotype monitoring for clinical trials: panels and methods from the ONE study.

BACKGROUND: Immune monitoring by flow cytometry is a fast and highly informative way of studying the effects of novel therapeutics aimed at reducing transplant rejection or treating autoimmune diseases. The ONE Study consortium has recently initiated a series of clinical trials aimed at using different cell therapies to promote tolerance to renal allografts. To compare the effectiveness of different cell therapies, the consortium developed a robust immune monitoring strategy, including procedures for whole blood (WB) leukocyte subset profiling by flow cytometry. METHODS: Six leukocyte profiling panels computing 7- to 9-surface marker antigens for monitoring the major leukocyte subsets as well as characteristics of T cell, B cell, and dendritic cell (DC) subsets were designed. The precision and variability of these panels were estimated. The assay was standardized within eight international laboratories using Flow-Set Pro beads for mean fluorescence intensity target definition and the flow cytometer setup procedure. Standardization was demonstrated by performing inter-site comparisons. RESULTS: Optimized methods for sample collection, storage, preparation, and analysis were established, including protocols for gating target subsets. WB specimen age testing demonstrated that staining must be performed within 4 hours of sample collection to keep variability low, meaning less than or equal to 10% for the majority of defined leukocyte subsets. Inter-site comparisons between all participating centers testing shipped normal WB revealed good precision, with a variability of 0.05% to 30% between sites. Intra-assay analyses revealed a variability of 0.05% to 20% for the majority of subpopulations. This was dependent on the frequency of the particular subset, with smaller subsets showing higher variability. The intra-assay variability performance defined limits of quantitation (LoQ) for subsets, which will be the basis for assessing statistically significant differences achieved by the different cell therapies. CONCLUSIONS: Local performance and central analysis of the ONE Study flow cytometry panel yields acceptable variability in a standardized assay at multiple international sites. These panels and procedures with WB allow unmanipulated analysis of changes in absolute cell numbers of leukocyte subsets in single- or multicenter clinical trials. Accordingly, we propose the ONE Study panel may be adopted as a standardized method for monitoring patients in clinical trials enrolling transplant patients, particularly trials of novel tolerance promoting therapies, to facilitate fair and meaningful comparisons between trials.

Organ-specific cellular requirements for in vivo dendritic cell generation.

Bone marrow-derived dendritic cell (DC) precursors seed peripheral organs, where they encounter diverse cellular environments during their final differentiation into DCs. Flt3 ligand (Flt3-L) is critical for instructing DC generation throughout different organs. However, it remains unknown which cells produce Flt3-L and, importantly, which cellular source drives DC development in such a variety of organs. Using a novel BAC transgenic Flt3-L reporter mouse strain coexpressing enhanced GFP and luciferase, we show ubiquitous Flt3-L expression in organs and cell types. These results were further confirmed at the protein level. Although Flt3-L was produced by immune and nonimmune cells, the source required for development of the DC compartment clearly differed among organs. In lymphoid organs such as the spleen and bone marrow, Flt3-L production by hemopoietic cells was critical for generation of normal DC numbers. This was unexpected for the spleen because both immune and nonimmune cells equally contributed to the Flt3-L content in that organ. Thus, localized production rather than the total tissue content of Flt3-L in spleen dictated normal splenic DC development. No differences were observed in the number of DC precursors, suggesting that the immune source of Flt3-L promoted pre-cDC differentiation in spleen. In contrast, DC generation in the lung, kidney, and pancreas was mostly driven by nonhematopoietic cells producing Flt3-L, with little contribution by immune cells. These findings demonstrate a high degree of flexibility in Flt3-L-dependent DC generation to adapt this process to organ-specific cellular environments encountered by DC precursors during their final differentiation.

Review of murine dendritic cells: types, location, and development.

Dendritic cells (DCs) are key coordinators of the immune response, governing the choice between tolerance and immunity. DCs are professional antigen-presenting cells capable of presenting antigen on MHC molecules and priming CD4 and CD8 T-cell responses. They form a heterogeneous group of cells based on phenotype, location, and function. In this review, murine DCs will be discussed regarding their function with special emphasis on their tissue distribution. Recent findings on DC homeostasis during cancer progression will be presented. Finally, the developmental pathways leading to DC differentiation from their precursors will be summarized.

Homeostasis of dendritic cells in lymphoid organs is controlled by regulation of their precursors via a feedback loop.

Dendritic cells (DCs) are key coordinators of the immune response, governing the choice between tolerance and immunity. Despite their importance, the mechanisms controlling the size of the DC compartment are largely unknown. Using a mouse model allowing continuous DC depletion, we show that maintenance of DC numbers in spleen is an active process mediated by Flt3-L-dependent regulation of precursor differentiation into DCs, rather than by changes in proliferation of the differentiated DCs. In particular, the frequency and differentiation potential of intrasplenic DC precursors increased in response to reduced DC numbers. Levels of Flt3-L, a cytokine required for DC differentiation, increased in the blood after DC depletion and returned to normal levels once the DC compartment filled up again. Our data suggest a feedback regulation of DC homeostasis whereby reduction of the DC pool size promotes differentiation of their precursors, via increased Flt3-L availability. This mechanism is different to those known for other immune cell types, such as the B- and T-cell compartments, whereby lymphopenia induces proliferation of already differentiated lymphocytes.

Drug-inducible remote control of gene expression by probiotic Escherichia coli Nissle 1917 in intestine, tumor and gall bladder of mice.

The probiotic bacterium Escherichia coli Nissle 1917 (EcN) constitutes a prospective vector for delivering heterologous therapeutic molecules to treat several human disorders. To add versatility to this carrier system, bacteria should be equipped with expression modules that can be regulated deliberately in a temporal and quantitative manner. This approach is called in vivo remote control (IVRC) of bacterial vectors. Here, we have evaluated promoters P(araBAD), P(rhaBAD) and P(tet), which can be induced with L-arabinose, L-rhamnose or anhydrotetracycline, respectively. EcN harboring promoter constructs with luciferase as reporter gene were administered either orally to healthy mice or intravenously to tumor bearing animals. Subsequent to bacterial colonization of tissues, inducer substances were administered via the oral or systemic route. By use of in vivo bioluminescence imaging, the time course of reporter gene expression was analyzed. Each promoter displayed a specific in vivo induction profile depending on the niche of bacterial residence and the route of inducer administration. Importantly, we also observed colonization of gall bladders of mice when EcN was administered systemically at high doses. Bacteria in this anatomical compartment remained accessible to remote control of bacterial gene expression.

An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice.

We describe a transgenic mouse line, Pax8-rtTA, which, under control of the mouse Pax8 promoter, directs high levels of expression of the reverse tetracycline-dependent transactivator (rtTA) to all proximal and distal tubules and the entire collecting duct system of both embryonic and adult kidneys. Using crosses of Pax8-rtTA mice with tetracycline-responsive c-MYC mice, we established a new, inducible model of polycystic kidney disease that can mimic adult onset and that shows progression to renal malignant disease. When targeting the expression of transforming growth factor beta-1 to the kidney, we avoided early lethality by discontinuous treatment and successfully established an inducible model of renal fibrosis. Finally, a conditional knockout of the gene encoding tuberous sclerosis complex-1 was achieved, which resulted in the early outgrowth of giant polycystic kidneys reminiscent of autosomal recessive polycystic kidney disease. These experiments establish Pax8-rtTA mice as a powerful tool for modeling renal diseases in transgenic mice.

Quantitative comparison of click beetle and firefly luciferases for in vivo bioluminescence imaging.

For bioluminescence imaging (BLI) of small animals, the most commonly used luciferase is Fluc from the firefly, but recently, green (CBGr99) and red (CBRed) click beetle luciferases became available. Because signal attenuation by tissues is lower for red light, red luciferases appear to be advantageous for BLI, but this has not been thoroughly tested. We compare different luciferases for BLI. For this purpose, cell transfectants are generated expressing comparable amounts of CBGr99, CBRed, or Fluc. This is achieved by coexpression of the luciferase with eGFP using the bicistronic 2A system, which results in stoichiometric coexpression of the respective proteins. In vitro, the CBGr99 transfectant exhibits the strongest total photon yield. For in vivo BLI, the transfectants are injected into mice at different locations. At a subcutaneous position, CBGr99 is clearly superior to the other luciferases. When the tumor cells are located in the peritoneum or lung, where more absorption by tissue occurs, CBGr99 and CBRed transfected cells emit a comparable number of red photons and are superior to Fluc, but CBGr99 reaches the maximum of the light emission faster than CBRed. Thus, although CBGr99 emits mainly green light, the high yield of total and red photons makes it an excellent candidate for BLI.

Remote control of tumour-targeted Salmonella enterica serovar Typhimurium by the use of L-arabinose as inducer of bacterial gene expression in vivo.

We have used Salmonella enterica serovar Typhimurium (S. typhimurium) which are able to colonize tumours besides spleen and liver. Bacteria were equipped with constructs encoding green fluorescent protein or luciferase as reporters under control of the promoter PBAD that is inducible with L-arabinose. Reporter genes could be induced in culture but also when the bacteria resided within the mouse macrophages J774A.1. More important, strong expression of reporters by the bacteria could be detected in mice after administration of L-arabinose. This was especially pronounced in bacteria colonizing tumours. Histology demonstrated that the bacteria had accumulated in and close to necrotic areas of tumours. Bacterial gene induction was observed in both regions. PBAD is tightly controlled also in vivo because gene E of bacteriophage PhiX174 could be introduced as inducible suicide gene. The possibility to deliberately induce genes in bacterial carriers within the host should render them extremely powerful tools for tumour therapy.