Tissue resident iNKT17 cells facilitate cancer cell extravasation in liver metastasis via interleukin-22.

During metastasis, cancer cells invade, intravasate, enter the circulation, extravasate, and colonize target organs. Here, we examined the role of interleukin (IL)-22 in metastasis. Immune cell-derived IL-22 acts on epithelial tissues, promoting regeneration and healing upon tissue damage, but it is also associated with malignancy. Il22-deficient mice and mice treated with an IL-22 antibody were protected from colon-cancer-derived liver and lung metastasis formation, while overexpression of IL-22 promoted metastasis. Mechanistically, IL-22 acted on endothelial cells, promoting endothelial permeability and cancer cell transmigration via induction of endothelial aminopeptidase N. Multi-parameter flow cytometry and single-cell sequencing of immune cells isolated during cancer cell extravasation into the liver revealed iNKT17 cells as source of IL-22. iNKT-cell-deficient mice exhibited reduced metastases, which was reversed by injection of wild type, but not Il22-deficient, invariant natural killer T (iNKT) cells. IL-22-producing iNKT cells promoting metastasis were tissue resident, as demonstrated by parabiosis. Thus, IL-22 may present a therapeutic target for prevention of metastasis.

Tr1 Cells Emerge and Suppress Effector Th17 Cells in Glomerulonephritis.

T regulatory type 1 (Tr1) cells, which are defined by their regulatory function, lack of Foxp3, and high expression of IL-10, CD49b, and LAG-3, are known to be able to suppress Th1 and Th17 in the intestine. Th1 and Th17 cells are also the main drivers of crescentic glomerulonephritis (GN), the most severe form of renal autoimmune disease. However, whether Tr1 cells emerge in renal inflammation and, moreover, whether they exhibit regulatory function during GN have not been thoroughly investigated yet. To address these questions, we used a mouse model of experimental crescentic GN and double Foxp3mRFP IL-10eGFP reporter mice. We found that Foxp3neg IL-10-producing CD4+ T cells infiltrate the kidneys during GN progression. Using single-cell RNA sequencing, we could show that these cells express the core transcriptional factors characteristic of Tr1 cells. In line with this, Tr1 cells showed a strong suppressive activity ex vivo and were protective in experimental crescentic GN in vivo. Finally, we could also identify Tr1 cells in the kidneys of patients with antineutrophil cytoplasmic autoantibody-associated GN and define their transcriptional profile. Tr1 cells are currently used in several immune-mediated inflammatory diseases, such as T-cell therapy. Thus, our study provides proof of concept for Tr1 cell-based therapies in experimental GN.

Differences in healthy lifestyle habits in university students as a function of academic area.

BACKGROUND: It is known that the promotion and acquisition of healthy attitudes is a key factor depending on the academic training provided by the university studies on which students are enrolled.The aim of the present research is to analyse and compare lifestyle habits and health-related quality of life (HRQoL) as a function of academic training. METHODS: A cross-sectional study with a volunteer convenience sample of 707 undergraduate participants aged (21.98 ± 3.50 years). Students were divided into four different groups according to their area of academic training. Socio-demographic variables, adherence to the Mediterranean diet (MD), physical activity (PA) engagement and HRQoL were recorded. RESULTS: PA and sport science students reported better scores in PA (6342.39 ± 2313.99 metabolic equivalents [METs]; P = 0.000), MD adherence (6.33 ± 2.69; P = 0.000) and HRQoL in physical and mental health (MH; 54.85 ± 9.18; 53.70 ± 13.6; P = 0.000). In contrast, non-health-related sciences (NHRS) students reported the lowest scores on assessed items. Students with a medium/high monthly salary reported better MD adherence (6.16 ± 3.07; P = 0.012). In addition, females reported better scores (6.41 ± 2.65; P = 0.000) than males. Further, males indicated better perceptions of MH (46.52 ± 18.84; P = 0.014). Moreover, university students with a high level of MD adherence were revealed to engage in more PA (5181.17 ± 2813.35 METs; P = 0.000) and have better HRQoL with regards to both physical (54.76 ± 8.84; P = 0.000) and MH (48.11 ± 16.73; P = 0.000). CONCLUSIONS: Outcomes point to differences in MD adherence, PA and HRQoL according to academic training. NHRS students who did not know healthy habits reported lower scores for all studied items. This indicates the need for health interventions at universities.

IL22BP Mediates the Antitumor Effects of Lymphotoxin Against Colorectal Tumors in Mice and Humans.

BACKGROUND & AIMS: Unregulated activity of interleukin (IL) 22 promotes intestinal tumorigenesis in mice. IL22 binds the antagonist IL22 subunit alpha 2 (IL22RA2, also called IL22BP). We studied whether alterations in IL22BP contribute to colorectal carcinogenesis in humans and mice. METHODS: We obtained tumor and nontumor tissues from patients with colorectal cancer (CRC) and measured levels of cytokines by quantitative polymerase chain reaction, flow cytometry, and immunohistochemistry. We measured levels of Il22bp messenger RNA in colon tissues from wild-type, Tnf-/-, Lta-/-, and Ltb-/- mice. Mice were given azoxymethane and dextran sodium sulfate to induce colitis and associated cancer or intracecal injections of MC38 tumor cells. Some mice were given inhibitors of lymphotoxin beta receptor (LTBR). Intestine tissues were analyzed by single-cell sequencing to identify cell sources of lymphotoxin. We performed immunohistochemistry analysis of colon tissue microarrays from patients with CRC (1475 tissue cores, contained tumor and nontumor tissues) and correlated levels of IL22BP with patient survival times. RESULTS: Levels of IL22BP were decreased in human colorectal tumors, compared with nontumor tissues, and correlated with levels of lymphotoxin. LTBR signaling was required for expression of IL22BP in colon tissues of mice. Wild-type mice given LTBR inhibitors had an increased tumor burden in both models, but LTBR inhibitors did not increase tumor growth in Il22bp-/- mice. Lymphotoxin directly induced expression of IL22BP in cultured human monocyte-derived dendritic cells via activation of nuclear factor κB. Reduced levels of IL22BP in colorectal tumor tissues were associated with shorter survival times of patients with CRC. CONCLUSIONS: Lymphotoxin signaling regulates expression of IL22BP in colon; levels of IL22BP are reduced in human colorectal tumors, associated with shorter survival times. LTBR signaling regulates expression of IL22BP in colon tumors in mice and cultured human dendritic cells. Patients with colorectal tumors that express low levels of IL22BP might benefit from treatment with an IL22 antagonist.

ImSpectR - R package to quantify immune repertoire diversity in spectratype and repertoire sequencing data.

SUMMARY: An effective immune system is characterized by a diverse immune repertoire. There is a strong demand for accurate and quantitative methods to assess the diversity of the immune repertoire for various (pre-)clinical applications, including the diagnosis and prognosis of primary immune deficiencies, or to assess the response to therapy. Current strategies for immune diversity assessment generally comprise the visual inspection of the length distribution of rearranged T- and B-cell receptors. Visual inspections, however, are prone to subjective assessments and thus lead to biases. Here, we introduce ImSpectR, a unified approach to quantify immunodiversity using either spectratype, repertoire sequencing or single cell RNA sequencing data. ImSpectR scores various types of deviations from the expected length distribution and integrates these into one measure, allowing for robust quantitative comparisons of immune diversity across individuals or conditions. AVAILABILITY: R-package is available for download on GitHub at https://github.com/martijn-cordes/ImSpectR. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Activation of the LMO2 oncogene through a somatically acquired neomorphic promoter in T-cell acute lymphoblastic leukemia.

Somatic mutations within noncoding genomic regions that aberrantly activate oncogenes have remained poorly characterized. Here we describe recurrent activating intronic mutations of LMO2, a prominent oncogene in T-cell acute lymphoblastic leukemia (T-ALL). Heterozygous mutations were identified in PF-382 and DU.528 T-ALL cell lines in addition to 3.7% of pediatric (6 of 160) and 5.5% of adult (9 of 163) T-ALL patient samples. The majority of indels harbor putative de novo MYB, ETS1, or RUNX1 consensus binding sites. Analysis of 5'-capped RNA transcripts in mutant cell lines identified the usage of an intermediate promoter site, with consequential monoallelic LMO2 overexpression. CRISPR/Cas9-mediated disruption of the mutant allele in PF-382 cells markedly downregulated LMO2 expression, establishing clear causality between the mutation and oncogene dysregulation. Furthermore, the spectrum of CRISPR/Cas9-derived mutations provides important insights into the interconnected contributions of functional transcription factor binding. Finally, these mutations occur in the same intron as retroviral integration sites in gene therapy-induced T-ALL, suggesting that such events occur at preferential sites in the noncoding genome.

Engineered T cells from induced pluripotent stem cells: from research towards clinical implementation.

Induced pluripotent stem cell (iPSC)-derived T (iT) cells represent a groundbreaking frontier in adoptive cell therapies with engineered T cells, poised to overcome pivotal limitations associated with conventional manufacturing methods. iPSCs offer an off-the-shelf source of therapeutic T cells with the potential for infinite expansion and straightforward genetic manipulation to ensure hypo-immunogenicity and introduce specific therapeutic functions, such as antigen specificity through a chimeric antigen receptor (CAR). Importantly, genetic engineering of iPSC offers the benefit of generating fully modified clonal lines that are amenable to rigorous safety assessments. Critical to harnessing the potential of iT cells is the development of a robust and clinically compatible production process. Current protocols for genetic engineering as well as differentiation protocols designed to mirror human hematopoiesis and T cell development, vary in efficiency and often contain non-compliant components, thereby rendering them unsuitable for clinical implementation. This comprehensive review centers on the remarkable progress made over the last decade in generating functional engineered T cells from iPSCs. Emphasis is placed on alignment with good manufacturing practice (GMP) standards, scalability, safety measures and quality controls, which constitute the fundamental prerequisites for clinical application. In conclusion, the focus on iPSC as a source promises standardized, scalable, clinically relevant, and potentially safer production of engineered T cells. This groundbreaking approach holds the potential to extend hope to a broader spectrum of patients and diseases, leading in a new era in adoptive T cell therapy.

A randomized, double-blind study on the efficacy of oral domperidone versus placebo for reducing SARS-CoV-2 viral load in mild-to-moderate COVID-19 patients in primary health care.

INTRODUCTION: The clinical effect of domperidone against COVID-19 has been investigated in a double-blind phase III clinical trial (EudraCT number 2021-001228-17). Domperidone has shown in vitro antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and potential immudolatory properties through the stimulation of prolactin secretion. PATIENTS AND METHODS: The efficacy of oral domperidone plus standard of care (SOC; n = 87) versus placebo plus SOC (n = 86) was evaluated in a 28-day randomized double-blind multicentre study in primary health care centres. A total of 173 outpatients with mild-to-moderate COVID-19 were included. Three daily doses of 10 mg (30 mg/day) of domperidone or placebo were administered for 7 days. Reduction of viral load on day 4 was the primary efficay endpoint. It was estimated in saliva samples by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), as the cycle thresholds detected ORF1ab, N Protein and S Protein genes. RESULTS: A significant reduction in the viral load was observed (p < 0.001) from baseline to days 4, 7 and 14 of the three genes studied with non-significant differences between domperidone and placebo groups. Twenty-three patients (13.3%) experienced adverse events, 14 patients in the domperidone group (16.1%) and 9 patients in the placebo group (10.5%). No patients needed to be hospitalized. CONCLUSION: Results do not prove the use of domperidone as antiviral in patients with COVID-19.

A 28-day double-blind clinical trial was performed to investigate the antiviral effect of domperidone, 30 mg/day for 7 days (n = 87) versus placebo (n = 86) in outpatients with mild-to-moderate COVID-19.The primary efficacy endpoint was the reduction of viral load on day 4 as compared with baseline, estimated as the cycle thresholds to detect ORF1ab, N Protein and S Protein genes by RT-qPCR in saliva samples.The study findings do not prove the use of domperidone as antiviral in patients with COVID-19.

Prevalence of aortic coarctation in Bogotá-Colombia from 2001 to 2018. The diagnostic needs to improve. / Prevalencia de coartación aórtica en Bogotá-Colombia de 2001 a 2018. El diagnóstico debe mejorar

OBJECTIVES: Aortic coarctation is the most frequent structural anomaly out of congenital heart diseases. This congenital defect is an important cause of death worldwide. We sought to determine the prevalence of aortic coarctation in Colombia and whether new policies have had an impact on its diagnosis. METHODS: In this study information from the Bogotá birth defect surveillance program during the years 2001-2018 from 63 hospitals was used. 537,026 live births of any weight and stillbirths of any weight were analyzed. The information was stored in a database on the servers of the Health Secretariat and the Pontificia Universidad Javeriana. We analyzed the presence of aortic coarctation according to the newborn's sex, weight, size, mother's age, and gestational age at the time of birth and when coarctation is accompanied by other types of congenital malformations. RESULTS: The prevalence of aortic coarctation in Bogotá during the years 2001-2018 found in this study was 1.25 in 10,000 live births. We also found that prevalence of aortic coarctation in Bogotá changes throughout the years having a significant increase in the year 2018 with 6.57 cases in 10,000 live births. CONCLUSIONS: This prevalence is higher than the one found in a study with data from 2001 to 2014, which suggests an improvement in the country's epidemiological surveillance and medical training. However, the prevalence found in Bogotá is still lower compared to the prevalence worldwide and from other continents, the prevalence for Latinamerica was significantly lower as compared to those in Asia, Europe, and United States so we emphasize the importance of continuing with improvements, such as standardizing screening methods and sensitivity of said methods in a local scale as well as a continental scale.

OBJETIVOS: La coartación aórtica es la anomalía estructural más frecuente de las cardiopatías congénitas. Este defecto congénito es una causa importante de muerte en todo el mundo. Buscamos determinar la prevalencia de la coartación aórtica en Colombia y si las nuevas políticas han tenido un impacto en su diagnóstico. MÉTODOS: En este estudio se utilizó información del programa de vigilancia de defectos de nacimiento de Bogotá durante los años 2001-2018 en 63 hospitales. Se analizaron 537,026 nacidos vivos de cualquier peso y nacidos muertos de cualquier peso. La información fue almacenada en una base de datos de los servidores de la Secretaría de Salud y de la Pontificia Universidad Javeriana. Se analizó la presencia de coartación aórtica de acuerdo al sexo del recién nacido, peso, tamaño, edad de la madre y edad gestacional en el momento del nacimiento y cuando la coartación se acompañó de otros tipos de malformaciones congénitas. RESULTADOS: La prevalencia de la coartación aórtica en Bogotá durante los años 2001-2018 encontrados en este estudio fue de 1.25 en 10,000 nacidos vivos. También encontramos que la prevalencia de coartación aórtica en Bogotá ha cambiado a lo largo de los años, teniendo un aumento significativo en el año 2018 con 6.57 casos en 10,000 nacidos vivos. CONCLUSIONES: Esta prevalencia es mayor que la encontrada en un estudio con datos de 2001 a 2014, lo que sugiere una mejora en la vigilancia epidemiológica y la formación médica del país. Si bien, la prevalencia encontrada en Bogotá es menor en comparación con la prevalencia a nivel mundial y de otros continentes, la prevalencia para el continente de Latinoamérica también es significativamente menor con respecto a Asia, Europa y Estados Unidos, por lo que enfatizamos la importancia de continuar con las mejoras, como la estandarización de los métodos de detección y la sensibilidad de dichos métodos tanto a nivel local como a nivel del continente.

Axin2/Conductin Is Required for Normal Haematopoiesis and T Lymphopoiesis.

The development of T lymphocytes in the thymus and their stem cell precursors in the bone marrow is controlled by Wnt signaling in strictly regulated, cell-type specific dosages. In this study, we investigated levels of canonical Wnt signaling during hematopoiesis and T cell development within the Axin2-mTurquoise2 reporter. We demonstrate active Wnt signaling in hematopoietic stem cells (HSCs) and early thymocytes, but also in more mature thymic subsets and peripheral T lymphocytes. Thymic epithelial cells displayed particularly high Wnt signaling, suggesting an interesting crosstalk between thymocytes and thymic epithelial cells (TECs). Additionally, reporter mice allowed us to investigate the loss of Axin2 function, demonstrating decreased HSC repopulation upon transplantation and the partial arrest of early thymocyte development in Axin2Tg/Tg full mutant mice. Mechanistically, loss of Axin2 leads to supraphysiological Wnt levels that disrupt HSC differentiation and thymocyte development.

Single-cell immune profiling reveals thymus-seeding populations, T cell commitment, and multilineage development in the human thymus.

T cell development in the mouse thymus has been studied extensively, but less is known regarding T cell development in the human thymus. We used a combination of single-cell techniques and functional assays to perform deep immune profiling of human T cell development, focusing on the initial stages of prelineage commitment. We identified three thymus-seeding progenitor populations that also have counterparts in the bone marrow. In addition, we found that the human thymus physiologically supports the development of monocytes, dendritic cells, and NK cells, as well as limited development of B cells. These results are an important step toward monitoring and guiding regenerative therapies in patients after hematopoietic stem cell transplantation.

Influence of Confinement Due to COVID-19 on Physical Activity and Mediterranean Diet Adherence and Its Relationship with Self-Esteem in Pre-Adolescent Students.

Regular physical activity and good adherence to the Mediterranean diet are important for improving physical and mental health. Confinement due to the COVID-19 pandemic has led to a lack of exercise and poor nutrition. Preadolescent mental health, specifically regarding self-esteem, may have been affected. This is particularly relevant between the ages of 10 and 14. The influence of total confinement due to COVID-19 on physical activity and Mediterranean diet adherence, and its relationship with self-esteem was studied in third-year primary school students and first-year secondary school students in the provinces of Granada and Malaga, Spain. Validated questionnaires were administered to evaluate physical activity (PAQ-C), Mediterranean diet adherence (KIDMED) and self-esteem (Rosenberg Scale). In the same way, the FAS III test was used to evaluate socioeconomic status and an ad-hoc questionnaire was developed to collect sociodemographic data and evaluate screen time. Data were analyzed using IBM SPSS 25.0 statistical software. During the period of total confinement, statistically significant differences were found between examined variables, according to sex, school year, school type, socioeconomic status and whether or not the participant had contracted COVID-19. These data were compared with those collected during a previous time period. Those with a low socioeconomic status and girls were most affected.

Combining Mobilizing Agents with Busulfan to Reduce Chemotherapy-Based Conditioning for Hematopoietic Stem Cell Transplantation.

In the context of hematopoietic stem cell (HSC) transplantation, conditioning with myelo- and immune-ablative agents is used to eradicate the patient's diseased cells, generate space in the marrow and suppress immune reactions prior to the infusion of donor HSCs. While conditioning is required for effective and long-lasting HSC engraftment, currently used regimens are also associated with short and long-term side effects on extramedullary tissues and even mortality. Particularly in patients with severe combined immunodeficiency (SCID), who are generally less than 1-year old at the time of transplantation and often suffer from existing comorbidities. There is a pressing need for development of alternative, less toxic conditioning regimens. Hence, we here aimed to improve efficacy of currently used myeloablative protocols by combining busulfan with stem-cell niche-directed therapeutic agents (G-CSF or plerixafor) that are approved for clinical use in stem cell mobilization. T, B and myeloid cell recovery was analyzed in humanized NSG mice after different conditioning regimens. Increasing levels of human leukocyte chimerism were observed in a busulfan dose-dependent manner, showing comparable immune recovery as with total body irradiation in CD34-transplanted NSG mice. Notably, a better T cell reconstitution compared to TBI was observed after busulfan conditioning not only in NSG mice but also in SCID mouse models. Direct effects of reducing the stem cell compartment in the bone marrow were observed after G-CSF and plerixafor administration, as well as in combination with low doses of busulfan. Unfortunately, these direct effects on the stem population in the bone marrow were not reflected in increased human chimerism or immune recovery after CD34 transplantation in NSG mice. These results indicate moderate potential of reduced conditioning regimens for clinical use relevant for all allogeneic transplants.

Impact of COVID-19 pandemic on cardiology fellows academic training: beyond the clinical impact.

OBJETIVO: Determinar la percepción de los médicos internos residentes (MIR) de cardiología de España sobre el efecto de la pandemia por COVID-19 en su formación y la adaptación realizada por sus servicios. MÉTODOS: Estudio de corte transversal a través de una plataforma de encuesta digital con el objetivo de conocer la opinión individual de los MIR de cardiología sobre la influencia de la pandemia en su formación. Se realiza un análisis estadístico para determinar los factores que influyeron en la percepción de la formación afectada. RESULTADOS: Participó un total de 180 MIR de las 17 comunidades autónomas (CA). Los MIR de tercer año fueron los más afectados, junto con los que rotaban en imagen cardíaca. Los residentes de las CA con una prevalencia >5 casos/1,000 habitantes fueron los que mayor probabilidad tuvieron de ser desplazados de sus servicios. CONCLUSIONES: Según la opinión de los participantes, el efecto de la pandemia por COVID-19 en su formación fue más negativa en los residentes de tercer año y los que rotaban en imagen cardíaca. OBJECTIVE: The objectives were to analyze the perception of the Cardiology Fellows in Training (FIT) of Spain about the impact of the COVID-19 pandemic on their academic training and to know the adaptative changes performed by their department. METHODS: A cross-sectional study performed through a digital survey platform for Cardiology FIT. Chi2 analysis and logistic regression were performed to determine the factors that influenced on the perception of an affected training. RESULTS: A total of 180 FIT from the 17 regions of Spain participated. Third year FIT and those rotating in cardiac imaging were the most affected with statistically significant difference. The residents of the regions with a prevalence of >5 cases/1,000 inhabitants were the most likely to be displaced from their departments. CONCLUSIONS: According to the opinion of the participants, the impact of the COVID-19 pandemic on their academic training was more negative in third year FITs and those rotating in cardiac imaging.

Infective endocarditis in patients with solid organ transplantation. A nationwide descriptive study.

BACKGROUND: Solid organ transplantation (SOT) implies immunosuppression and frequent health care contact. Our aim was to compare the characteristics of patients with infective endocarditis (IE) and SOT against those without SOT. METHODS: We used data from the Spanish Collaboration on Endocarditis during the period 2008-2018. RESULTS: We identified 4794 cases of IE, 85 (1.8%) in SOT (56 kidney, 18 liver, 8 heart, 3 lung). Thirteen patients with other transplantation types (bone marrow, hematopoietic precursors, and cornea) were excluded from the analysis. Compared with patients without SOT, patients with SOT had lower median age (61 vs. 69 years, p<0.001), more comorbidities (mean age-adjusted Charlson index 5.7±2.9 vs. 4.9±2.9, p=0.004), a lower prevalence of native valvular heart disease (29.4 vs. 45.4%, p=0.003), more in-hospital and healthcare-related IE (70.5% vs. 36.3%, p<0.001) and staphylococcal etiology (57.7% vs. 39.7%, p=0.001). Patients with SOT had more frequent kidney function worsening (47.1% vs. 34.6%, p=0.02), septic shock (25.9% vs. 12.1 %, p<0.001), sepsis (27.1% vs. 17.2%, p=0.02), and less surgery indication (54.1% vs 66.3%, p=0.02) and surgery (32.9% vs. 46.3%, p=0.01) than patients without SOT. There were no significant differences in mortality: inhospital (30.6% SOT vs. 25.6% without SOT, p=0.31), 1-year (38.8% SOT vs. 31.9% without SOT, p=0.18). CONCLUSIONS: Most IE in SOT recipients are nosocomial and over 70% are health care-related. Half have previously normal heart valves and almost 60% are due to Staphylococcus spp. infections. Mortality seems to be similar to non-SOT counterparts.

Preclinical Development of Autologous Hematopoietic Stem Cell-Based Gene Therapy for Immune Deficiencies: A Journey from Mouse Cage to Bed Side.

Recent clinical trials using patient's own corrected hematopoietic stem cells (HSCs), such as for primary immunodeficiencies (Adenosine deaminase (ADA) deficiency, X-linked Severe Combined Immunodeficiency (SCID), X-linked chronic granulomatous disease (CGD), Wiskott-Aldrich Syndrome (WAS)), have yielded promising results in the clinic; endorsing gene therapy to become standard therapy for a number of diseases. However, the journey to achieve such a successful therapy is not easy, and several challenges have to be overcome. In this review, we will address several different challenges in the development of gene therapy for immune deficiencies using our own experience with Recombinase-activating gene 1 (RAG1) SCID as an example. We will discuss product development (targeting of the therapeutic cells and choice of a suitable vector and delivery method), the proof-of-concept (in vitro and in vivo efficacy, toxicology, and safety), and the final release steps to the clinic (scaling up, good manufacturing practice (GMP) procedures/protocols and regulatory hurdles).

Functional definition of a transcription factor hierarchy regulating T cell lineage commitment.

T cell factor 1 (Tcf1) is the first T cell-specific protein induced by Notch signaling in the thymus, leading to the activation of two major target genes, Gata3 and Bcl11b. Tcf1 deficiency results in partial arrests in T cell development, high apoptosis, and increased development of B and myeloid cells. Phenotypically, seemingly fully T cell-committed thymocytes with Tcf1 deficiency have promiscuous gene expression and an altered epigenetic profile and can dedifferentiate into more immature thymocytes and non-T cells. Restoring Bcl11b expression in Tcf1-deficient cells rescues T cell development but does not strongly suppress the development of non-T cells; in contrast, expressing Gata3 suppresses their development but does not rescue T cell development. Thus, T cell development is controlled by a minimal transcription factor network involving Notch signaling, Tcf1, and the subsequent division of labor between Bcl11b and Gata3, thereby ensuring a properly regulated T cell gene expression program.

A Novel Branched DNA-Based Flowcytometric Method for Single-Cell Characterization of Gene Therapy Products and Expression of Therapeutic Genes.

Many preclinical and clinical studies of hematopoietic stem cell-based gene therapy (GT) are based on the use of lentiviruses as the vector of choice. Assessment of the vector titer and transduction efficiency of the cell product is critical for these studies. Efficacy and safety of the modified cell product are commonly determined by assessing the vector copy number (VCN) using qPCR. However, this optimized and well-established method in the GT field is based on bulk population averages, which can lead to misinterpretation of the actual VCN per transduced cell. Therefore, we introduce here a single cell-based method that allows to unmask cellular heterogeneity in the GT product, even when antibodies are not available. We use Invitrogen's flow cytometry-based PrimeFlow™ RNA Assay with customized probes to determine transduction efficiency of transgenes of interest, promoter strength, and the cellular heterogeneity of murine and human stem cells. The assay has good specificity and sensitivity to detect the transgenes, as shown by the high correlations between PrimeFlow™-positive cells and the VCN. Differences in promoter strengths can readily be detected by differences in percentages and fluorescence intensity. Hence, we show a customizable method that allows to determine the number of transduced cells and the actual VCN per transduced cell in a GT product. The assay is suitable for all therapeutic genes for which antibodies are not available or too cumbersome for routine flow cytometry. The method also allows co-staining of surface markers to analyze differential transduction efficiencies in subpopulations of target cells.

Successful Preclinical Development of Gene Therapy for Recombinase-Activating Gene-1-Deficient SCID.

Recombinase-activating gene-1 (RAG1)-deficient severe combined immunodeficiency (SCID) patients lack B and T lymphocytes due to the inability to rearrange immunoglobulin and T cell receptor genes. Gene therapy is an alternative for those RAG1-SCID patients who lack a suitable bone marrow donor. We designed lentiviral vectors with different internal promoters driving codon-optimized RAG1 to ensure optimal expression. We used Rag1 -/- mice as a preclinical model for RAG1-SCID to assess the efficacy of the various vectors. We observed that B and T cell reconstitution directly correlated with RAG1 expression. Mice with low RAG1 expression showed poor immune reconstitution; however, higher expression resulted in phenotypic and functional lymphocyte reconstitution comparable to mice receiving wild-type stem cells. No signs of genotoxicity were found. Additionally, RAG1-SCID patient CD34+ cells transduced with our clinical RAG1 vector and transplanted into NSG mice led to improved human B and T cell development. Considering this efficacy outcome, together with favorable safety data, these results substantiate the need for a clinical trial for RAG1-SCID.