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  1. Article ; Online: Point-of-care cell therapy manufacturing; it’s not for everyone

    David F. Stroncek / Robert P. T. Somerville / Steven L. Highfill

    Journal of Translational Medicine, Vol 20, Iss 1, Pp 1-

    2022  Volume 3

    Abstract: Abstract The use of cellular therapies to treat cancer, inherited immune deficiencies, hemoglobinopathies and viral infections is growing rapidly. The increased interest in cellular therapies has led to the development of reagents and closed-system ... ...

    Abstract Abstract The use of cellular therapies to treat cancer, inherited immune deficiencies, hemoglobinopathies and viral infections is growing rapidly. The increased interest in cellular therapies has led to the development of reagents and closed-system automated instruments for the production of these therapies. For cellular therapy clinical trials involving multiple sites some people are advocating a decentralized model of manufacturing where patients are treated with cells produced using automated instruments at each participating center using a single, centrally held Investigational New Drug Application (IND). Many academic centers are purchasing these automated instruments for point-of-care manufacturing and participation in decentralized multiple center clinical trials. However, multiple site manufacturing requires harmonization of product testing and manufacturing in order to interpret the clinical trial results. Decentralized manufacturing is quite challenging since all centers should use the same manufacturing protocol, the same or comparable in-process and lot release assays and the quality programs from each center must work closely together. Consequently, manufacturing cellular therapies using a decentralized model is in many ways more difficult than manufacturing cells in a single centralized facility. Before an academic center decides to establish a point-of-care cell processing laboratory, they should consider all costs associated with such a program. For many academic cell processing centers, point-of-care manufacturing may not be a good investment.
    Keywords Cell therapy ; Gene therapy ; Cancer immunotherapy ; Point-of-care manufacturing ; Decentralized manufacturing ; Medicine ; R
    Subject code 629
    Language English
    Publishing date 2022-01-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Immunomagnetic B cell isolation as a tool to study blood cell subsets and enrich B cell transcripts

    Amanda N. Henning / Daniel Green / Ryan Baumann / Patrick Grandinetti / Steven L. Highfill / Huizhi Zhou / Valeria De Giorgi

    BMC Research Notes, Vol 14, Iss 1, Pp 1-

    2021  Volume 7

    Abstract: Abstract Objective Transcriptional profiling of immune cells is an indispensable tool in biomedical research; however, heterogenous sample types routinely used in transcriptomic studies may mask important cell type-specific transcriptional differences. ... ...

    Abstract Abstract Objective Transcriptional profiling of immune cells is an indispensable tool in biomedical research; however, heterogenous sample types routinely used in transcriptomic studies may mask important cell type-specific transcriptional differences. Techniques to isolate desired cell types are used to overcome this limitation. We sought to evaluate the use of immunomagnetic B cell isolation on RNA quality and transcriptional output. Additionally, we aimed to develop a B cell gene signature representative of a freshly isolated B cell population to be used as a tool to verify isolation efficacy and to provide a transcriptional standard for evaluating maintenance or deviation from traditional B cell identity. Results We found RNA quality and RNA-sequencing output to be comparable between donor-matched PBMC, whole blood, and B cells following negative selection by immunomagnetic B cell isolation. Transcriptional analysis enabled the development of an 85 gene B cell signature. This signature effectively clustered isolated B cells from heterogeneous sample types in our study and naïve and memory B cells when applied to transcriptional data from a published source. Additionally, by identifying B cell signature genes whose functional role in B cells is currently unknown, our gene signature has uncovered areas for future investigation.
    Keywords RNA-sequencing ; B cells ; Differential gene expression ; Gene signature ; Medicine ; R ; Biology (General) ; QH301-705.5 ; Science (General) ; Q1-390
    Language English
    Publishing date 2021-11-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: High efficiency closed-system gene transfer using automated spinoculation

    Victoria Ann Remley / Jianjian Jin / Sarmila Sarkar / Larry Moses / Michaela Prochazkova / Yihua Cai / Lipei Shao / Hui Liu / Tatyana Fuksenko / Ping Jin / David F. Stroncek / Steven L. Highfill

    Journal of Translational Medicine, Vol 19, Iss 1, Pp 1-

    2021  Volume 15

    Abstract: Abstract Background Gene transfer is an important tool for cellular therapies. Lentiviral vectors are most effectively transferred into lymphocytes or hematopoietic progenitor cells using spinoculation. To enable cGMP (current Good Manufacturing Practice) ...

    Abstract Abstract Background Gene transfer is an important tool for cellular therapies. Lentiviral vectors are most effectively transferred into lymphocytes or hematopoietic progenitor cells using spinoculation. To enable cGMP (current Good Manufacturing Practice)-compliant cell therapy production, we developed and compared a closed-system spinoculation method that uses cell culture bags, and an automated closed system spinoculation method to decrease technician hands on time and reduce the likelihood for microbial contamination. Methods Sepax spinoculation, bag spinoculation, and static bag transduction without spinoculation were compared for lentiviral gene transfer in lymphocytes collected by apheresis. The lymphocytes were transduced once and cultured for 9 days. The lentiviral vectors tested encoded a CD19/CD22 Bispecific Chimeric Antigen Receptor (CAR), a FGFR4-CAR, or a CD22-CAR. Sepax spinoculation times were evaluated by testing against bag spinoculation and static transduction to optimize the Sepax spin time. The Sepax spinoculation was then used to test the transduction of different CAR vectors. The performance of the process using healthy donor and a patient sample was evaluated. Functional assessment was performed of the CD19/22 and CD22 CAR T-cells using killing assays against the NALM6 tumor cell line and cytokine secretion analysis. Finally, gene expression of the transduced T-cells was examined to determine if there were any major changes that may have occurred as a result of the spinoculation process. Results The process of spinoculation lead to significant enhancement in gene transfer. Sepax spinoculation using a 1-h spin time showed comparable transduction efficiency to the bag spinoculation, and much greater than the static bag transduction method (83.4%, 72.8%, 35.7% n = 3). The performance of three different methods were consistent for all lentiviral vectors tested and no significant difference was observed when using starting cells from healthy donor versus a patient sample. Sepax spinoculation ...
    Keywords Sepax ; Spinoculation ; CAR T-cell ; Gene transfer ; Medicine ; R
    Subject code 500
    Language English
    Publishing date 2021-11-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCR T cell products

    Alex Lu / Hui Liu / Rongye Shi / Yihua Cai / Jinxia Ma / Lipei Shao / Victor Rong / Nikolaos Gkitsas / Hong Lei / Steven L. Highfill / Sandhya Panch / David F. Stroncek / Ping Jin

    Journal of Translational Medicine, Vol 18, Iss 1, Pp 1-

    2020  Volume 7

    Abstract: Abstract Background Genetically engineered T cells have become an important therapy for B-cell malignancies. Measuring the efficiency of vector integration into the T cell genome is important for assessing the potency and safety of these cancer ... ...

    Abstract Abstract Background Genetically engineered T cells have become an important therapy for B-cell malignancies. Measuring the efficiency of vector integration into the T cell genome is important for assessing the potency and safety of these cancer immunotherapies. Methods A digital droplet polymerase chain reaction (ddPCR) assay was developed and evaluated for assessing the average number of lenti- and retroviral vectors integrated into Chimeric Antigen Receptor (CAR) and T Cell Receptor (TCR)-engineered T cells. Results The ddPCR assay consistently measured the concentration of an empty vector in solution and the average number of CAR and TCR vectors integrated into T cell populations. There was a linear relationship between the average vector copy number per cell measured by ddPCR and the proportion of cells transduced as measured by flow cytometry. Similar vector copy number measurements were obtained by different staff using the ddPCR assay, highlighting the assays reproducibility among technicians. Analysis of fresh and cryopreserved CAR T and TCR engineered T cells yielded similar results. Conclusions ddPCR is a robust tool for accurate quantitation of average vector copy number in CAR and TCR engineered T cells. The assay is also applicable to other types of genetically engineered cells including Natural Killer cells and hematopoietic stem cells.
    Keywords Droplet digital PCR ; Vector copy number ; Genetically engineered T cells ; Gene therapy ; Chimeric antigen receptor (CAR) T cells ; T Cell Receptor (TCR)-engineered T cells ; Medicine ; R
    Subject code 570
    Language English
    Publishing date 2020-05-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: Genome-wide profiling of retroviral DNA integration and its effect on clinical pre-infusion CAR T-cell products

    Lipei Shao / Rongye Shi / Yingdong Zhao / Hui Liu / Alexander Lu / Jinxia Ma / Yihua Cai / Tatyana Fuksenko / Alejandra Pelayo / Nirali N. Shah / James N. Kochenderfer / Scott M. Norberg / Christian Hinrichs / Steven L. Highfill / Robert P Somerville / Sandhya R. Panch / Ping Jin / David F. Stroncek

    Journal of Translational Medicine, Vol 20, Iss 1, Pp 1-

    2022  Volume 15

    Abstract: Abstract Background Clinical CAR T-cell therapy using integrating vector systems represents a promising approach for the treatment of hematological malignancies. Lentiviral and γ-retroviral vectors are the most commonly used vectors in the manufacturing ... ...

    Abstract Abstract Background Clinical CAR T-cell therapy using integrating vector systems represents a promising approach for the treatment of hematological malignancies. Lentiviral and γ-retroviral vectors are the most commonly used vectors in the manufacturing process. However, the integration pattern of these viral vectors and subsequent effect on CAR T-cell products is still unclear. Methods We used a modified viral integration sites analysis (VISA) pipeline to evaluate viral integration events around the whole genome in pre-infusion CAR T-cell products. We compared the differences of integration pattern between lentiviral and γ-retroviral products. We also explored whether the integration sites correlated with clinical outcomes. Results We found that γ-retroviral vectors were more likely to insert than lentiviral vectors into promoter, untranslated, and exon regions, while lentiviral vector integration sites were more likely to occur in intron and intergenic regions. Some integration events affected gene expression at the transcriptional and post-transcriptional level. Moreover, γ-retroviral vectors showed a stronger impact on the host transcriptome. Analysis of individuals with different clinical outcomes revealed genes with differential enrichment of integration events. These genes may affect biological functions by interrupting amino acid sequences and generating abnormal proteins, instead of by affecting mRNA expression. These results suggest that vector integration is associated with CAR T-cell efficacy and clinical responses. Conclusion We found differences in integration patterns, insertion hotspots and effects on gene expression vary between lentiviral and γ-retroviral vectors used in CAR T-cell products and established a foundation upon which we can conduct further analyses.
    Keywords Medicine ; R
    Subject code 337
    Language English
    Publishing date 2022-11-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  6. Article ; Online: Single cell sequencing reveals gene expression signatures associated with bone marrow stromal cell subpopulations and time in culture

    Shutong Liu / David F. Stroncek / Yingdong Zhao / Victoria Chen / Rongye Shi / Jinguo Chen / Jiaqiang Ren / Hui Liu / Hee Joon Bae / Steven L. Highfill / Ping Jin

    Journal of Translational Medicine, Vol 17, Iss 1, Pp 1-

    2019  Volume 13

    Abstract: Abstract Background Bone marrow stromal cells (BMSCs) are a heterogeneous population that participates in wound healing, immune modulation and tissue regeneration. Next generation sequencing was used to analyze transcripts from single BMSCs in order to ... ...

    Abstract Abstract Background Bone marrow stromal cells (BMSCs) are a heterogeneous population that participates in wound healing, immune modulation and tissue regeneration. Next generation sequencing was used to analyze transcripts from single BMSCs in order to better characterize BMSC subpopulations. Methods Cryopreserved passage 2 BMSCs from one healthy subject were cultured through passage 10. The transcriptomes of bulk BMSCs from designated passages were analyzed with microarrays and RNA sequencing (RNA-Seq). For some passages, single BMSCs were separated using microfluidics and their transcriptomes were analyzed by RNA-Seq. Results Transcriptome analysis by microarray and RNA-Seq of unseparated BMSCs from passages 2, 4, 6, 8, 9 and 10 yielded similar results; both data sets grouped passages 4 and 6 and passages 9 and 10 together and genes differentially expressed among these early and late passage BMSCs were similar. 3D Diffusion map visualization of single BMSCs from passages 3, 4, 6, 8 and 9 clustered passages 3 and 9 into two distinct groups, but there was considerable overlap for passages 4, 6 and 8 cells. Markers for early passage, FGFR2, and late passage BMSCs, PLAT, were able to identify three subpopulations within passage 3 BMSCs; one that expressed high levels of FGFR2 and low levels of PLAT; one that expressed low levels of FGFR2 and high levels of PLAT and one that expressed intermediate levels of FGFR2 and low levels of PLAT. Conclusions Single BMSCs can be separated by microfluidics and their transcriptome analyzed by next generation sequencing. Single cell analysis of early passage BMSCs identified a subpopulation of cells expressing high levels of FGFR2 that might include skeletal stem cells.
    Keywords Bone marrow stromal cells ; Next generation sequencing ; Single cell next generation sequencing ; FGFR2 ; PLAT ; Medicine ; R
    Subject code 616
    Language English
    Publishing date 2019-01-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  7. Article ; Online: A Phase 1 trial of autologous monocytes stimulated ex vivo with Sylatron® (Peginterferon alfa-2b) and Actimmune® (Interferon gamma-1b) for intra-peritoneal administration in recurrent ovarian cancer

    Daniel S. Green / Ana T. Nunes / Virginia David-Ocampo / Irene B. Ekwede / Nicole D. Houston / Steven L. Highfill / Hanh Khuu / David F. Stroncek / Seth M. Steinberg / Kathryn C. Zoon / Christina M. Annunziata

    Journal of Translational Medicine, Vol 16, Iss 1, Pp 1-

    2018  Volume 9

    Abstract: Abstract Background Ovarian cancer has no definitive second line therapeutic options, and largely recurs in the peritoneal cavity. Locoregional immune therapy using both interferons and monocytes can be used as a novel approach. Interferons have both ... ...

    Abstract Abstract Background Ovarian cancer has no definitive second line therapeutic options, and largely recurs in the peritoneal cavity. Locoregional immune therapy using both interferons and monocytes can be used as a novel approach. Interferons have both cytostatic and cytotoxic properties, while monocytes stimulated with interferons have potent cytotoxic properties. Due to the highly immune suppressive properties of ovarian cancer, ex vivo stimulation of autologous patient monocytes with interferons and infusion of all three agents intraperitoneally (IP) can provide a strong pro-inflammatory environment at the site of disease to kill malignant cells. Methods Patient monocytes are isolated through counterflow elutriation and stimulated ex vivo with interferons and infused IP through a semi-permanent catheter. We have designed a standard 3 + 3 dose escalation study to explore the highest tolerated dose of interferons and monocytes infused IP in patients with chemotherapy resistant ovarian cancer. Secondary outcome measurements of changes in the peripheral blood immune compartment and plasma cytokines will be studied for correlations of response. Discussion We have developed a novel immunotherapy focused on the innate immune system for the treatment of ovarian cancer. We have combined the use of autologous monocytes and interferons alpha and gamma for local–regional administration directly into the peritoneal cavity. This therapy is highly unique in that it is the first study of its type using only components of the innate immune system for the locoregional delivery consisting of autologous monocytes and dual interferons alpha and gamma. Trial Registration ClinicalTrials.gov Identifier: NCT02948426, registered on October 28, 2016. https://clinicaltrials.gov/ct2/show/NCT02948426
    Keywords Cellular therapy ; Immunotherapy ; Monocytes ; Interferons ; Ovarian cancer ; Intraperitoneal ; Medicine ; R
    Subject code 610
    Language English
    Publishing date 2018-07-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  8. Article ; Online: Enhanced clinical-scale manufacturing of TCR transduced T-cells using closed culture system modules

    Jianjian Jin / Nikolaos Gkitsas / Vicki S. Fellowes / Jiaqiang Ren / Steven A. Feldman / Christian S. Hinrichs / David F. Stroncek / Steven L. Highfill

    Journal of Translational Medicine, Vol 16, Iss 1, Pp 1-

    2018  Volume 13

    Abstract: Abstract Background Genetic engineering of T-cells to express specific T cell receptors (TCR) has emerged as a novel strategy to treat various malignancies. More widespread utilization of these types of therapies has been somewhat constrained by the lack ...

    Abstract Abstract Background Genetic engineering of T-cells to express specific T cell receptors (TCR) has emerged as a novel strategy to treat various malignancies. More widespread utilization of these types of therapies has been somewhat constrained by the lack of closed culture processes capable of expanding sufficient numbers of T-cells for clinical application. Here, we evaluate a process for robust clinical grade manufacturing of TCR gene engineered T-cells. Methods TCRs that target human papillomavirus E6 and E7 were independently tested. A 21 day process was divided into a transduction phase (7 days) and a rapid expansion phase (14 days). This process was evaluated using two healthy donor samples and four samples obtained from patients with epithelial cancers. Results The process resulted in ~ 2000-fold increase in viable nucleated cells and high transduction efficiencies (64–92%). At the end of culture, functional assays demonstrated that these cells were potent and specific in their ability to kill tumor cells bearing target and secrete large quantities of interferon and tumor necrosis factor. Both phases of culture were contained within closed or semi-closed modules, which include automated density gradient separation and cell culture bags for the first phase and closed GREX culture devices and wash/concentrate systems for the second phase. Conclusion Large-scale manufacturing using modular systems and semi-automated devices resulted in highly functional clinical-grade TCR transduced T-cells. This process is now in use in actively accruing clinical trials and the NIH Clinical Center and can be utilized at other cell therapy manufacturing sites that wish to scale-up and optimize their processing using closed systems.
    Keywords E6 HPV ; E7 HPV ; HPV-16+ ; T-cell receptor ; Cellular therapy ; Cancer immunotherapy ; Medicine ; R
    Subject code 610
    Language English
    Publishing date 2018-01-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  9. Article ; Online: Production of a cellular product consisting of monocytes stimulated with Sylatron® (Peginterferon alfa-2b) and Actimmune® (Interferon gamma-1b) for human use

    Daniel S. Green / Ana T. Nunes / Kevin W. Tosh / Virginia David-Ocampo / Vicki S. Fellowes / Jiaqiang Ren / Jianjian Jin / Sue-Ellen Frodigh / Chauha Pham / Jolynn Procter / Celina Tran / Irene Ekwede / Hanh Khuu / David F. Stroncek / Steven L. Highfill / Kathryn C. Zoon / Christina M. Annunziata

    Journal of Translational Medicine, Vol 17, Iss 1, Pp 1-

    2019  Volume 13

    Abstract: Abstract Background Monocytes are myeloid cells that reside in the blood and bone marrow and respond to inflammation. At the site of inflammation, monocytes express cytokines and chemokines. Monocytes have been shown to be cytotoxic to tumor cells in the ...

    Abstract Abstract Background Monocytes are myeloid cells that reside in the blood and bone marrow and respond to inflammation. At the site of inflammation, monocytes express cytokines and chemokines. Monocytes have been shown to be cytotoxic to tumor cells in the presence of pro-inflammatory cytokines such as Interferon Alpha, Interferon Gamma, and IL-6. We have previously shown that monocytes stimulated with both interferons (IFNs) results in synergistic killing of ovarian cancer cells. We translated these observations to an ongoing clinical trial using adoptive cell transfer of autologous monocytes stimulated ex vivo with IFNs and infused into the peritoneal cavity of patients with advanced, chemotherapy resistant, ovarian cancer. Here we describe the optimization of the monocyte elutriation protocol and a cryopreservation protocol of the monocytes isolated from peripheral blood. Methods Counter flow elutriation was performed on healthy donors or women with ovarian cancer. The monocyte-containing, RO-fraction was assessed for total monocyte number, purity, viability, and cytotoxicity with and without a cryopreservation step. All five fractions obtained from the elutriation procedure were also assessed by flow cytometry to measure the percent of immune cell subsets in each fraction. Results Both iterative monocyte isolation using counter flow elutriation or cryopreservation following counter flow elutriation can yield over 2 billion monocytes for each donor with high purity. We also show that the monocytes are stable, viable, and retain cytotoxic functions when cultured with IFNs. Conclusion Large scale isolation of monocytes from both healthy donors and patients with advanced, chemotherapy resistant ovarian cancer, can be achieved with high total number of monocytes. These monocytes can be cryopreserved and maintain viability and cytotoxic function. All of the elutriated cell fractions contain ample immune cells which could be used for other cell therapy-based applications.
    Keywords Cell therapy ; Cellular immunotherapy ; Monocytes ; Interferons ; Innate immunity ; Medicine ; R
    Subject code 610
    Language English
    Publishing date 2019-03-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  10. Article ; Online: Nfatc2 and Tob1 have non-overlapping function in T cell negative regulation and tumorigenesis.

    Sarah L May / Qing Zhou / Mitzi Lewellen / Cristan M Carter / David Coffey / Steven L Highfill / Christoph M Bucher / Ilze Matise / Herbert C Morse / M Gerard O'Sullivan / Melissa Schutten / Charles Johnson / Donald Bellgrau / Bruce R Blazar / Jaime F Modiano

    PLoS ONE, Vol 9, Iss 6, p e

    2014  Volume 100629

    Abstract: Nfatc2 and Tob1 are intrinsic negative regulators of T cell activation. Nfatc2-deficient and Tob1-deficient T cells show reduced thresholds of activation; however, whether these factors have independent or overlapping roles in negative regulation of T ... ...

    Abstract Nfatc2 and Tob1 are intrinsic negative regulators of T cell activation. Nfatc2-deficient and Tob1-deficient T cells show reduced thresholds of activation; however, whether these factors have independent or overlapping roles in negative regulation of T cell responses has not been previously examined. Here, we show that Nfatc2 knockout (KO) but not Tob1 KO mice have age-associated accumulation of persistently activated T cells in vivo and expansion of the CD44+ memory cell compartment and age-associated lymphocytic infiltrates in visceral organs, without significant changes in numbers of CD4+CD25+Foxp3+ regulatory T cells (Treg). In vitro, CD4+CD25- "conventional" T cells (Tconvs) from both KO strains showed greater proliferation than wild type (WT) Tconvs. However, while Tregs from Nfatc2 KO mice retained normal suppressive function, Tregs from Tob1 KOs had enhanced suppressive activity. Nfatc2 KO Tconvs expanded somewhat more rapidly than WT Tconvs under conditions of homeostatic proliferation, but their accelerated growth capacity was negated, at least acutely, in a lymphoreplete environment. Finally, Nfatc2 KO mice developed a previously uncharacterized increase in B-cell malignancies, which was not accelerated by the absence of Tob1. The data thus support the prevailing hypothesis that Nfatc2 and Tob1 are non-redundant regulators of lymphocyte homeostasis.
    Keywords Medicine ; R ; Science ; Q
    Subject code 570
    Language English
    Publishing date 2014-01-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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