Publications by authors named "David Rawlings"

173 Publications

The Autoimmune Risk R262W Variant of the Adaptor SH2B3 Improves Survival in Sepsis.

J Immunol 2021 12 5;207(11):2710-2719. Epub 2021 Nov 5.

Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA;

The single-nucleotide polymorphism (SNP) rs3184504 is broadly associated with increased risk for multiple autoimmune and cardiovascular diseases. Although the allele is uniquely enriched in European descent, the mechanism for the widespread selective sweep is not clear. In this study, we find the rs3184504*T allele had a strong association with reduced mortality in a human sepsis cohort. The rs3184504*T allele associates with a loss-of-function amino acid change (p.R262W) in the adaptor protein SH2B3, a likely causal variant. To better understand the role of SH2B3 in sepsis, we used mouse modeling and challenged SH2B3-deficient mice with a polymicrobial cecal-ligation puncture (CLP) procedure. We found SH2B3 deficiency improved survival and morbidity with less organ damage and earlier bacterial clearance compared with control mice. The peritoneal infiltrating cells exhibited augmented phagocytosis in mice with enriched recruitment of Ly6C inflammatory monocytes despite equivalent or reduced chemokine expression. Rapid cycling of monocytes and progenitors occurred uniquely in the mice following CLP, suggesting augmented myelopoiesis. To model the hypomorphic autoimmune risk allele, we created a novel knockin mouse harboring a similar point mutation in the murine pleckstrin homology domain of SH2B3. At baseline, phenotypic changes suggested a hypomorphic allele. In the CLP model, homozygous knockin mice displayed improved mortality and morbidity compared with wild-type or heterozygous mice. Collectively, these data suggest that hypomorphic SH2B3 improves the sepsis response and that balancing selection likely contributed to the relative frequency of the autoimmune risk variant.
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http://dx.doi.org/10.4049/jimmunol.2100454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8612972PMC
December 2021

Cutting Edge: A Threshold of B Cell Costimulatory Signals Is Required for Spontaneous Germinal Center Formation in Autoimmunity.

J Immunol 2021 11 29;207(9):2217-2222. Epub 2021 Sep 29.

Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA;

Cognate interactions between autoreactive B and T cells promote systemic lupus erythematosus pathogenesis by inter alia facilitating spontaneous germinal center (GC) formation. Whereas both myeloid and B cell APCs express B7 ligands (CD80 and CD86), the prevailing model holds that dendritic cell costimulation is sufficient for CD28-dependent T cell activation. In this study, we report that B cell-intrinsic CD80/CD86 deletion unexpectedly abrogates GCs in murine lupus. Interestingly, absent GCs differentially impacted serum autoantibodies. In keeping with distinct extrafollicular and GC activation pathways driving lupus autoantibodies, lack of GCs correlated with loss of RNA-associated autoantibodies but preserved anti-dsDNA and connective tissue autoantibody titers. Strikingly, even heterozygous B cell CD80/CD86 deletion was sufficient to prevent autoimmune GCs and RNA-associated autoantibodies. Together, these findings identify a key mechanism whereby B cells promote lupus pathogenesis by providing a threshold of costimulatory signals required for autoreactive T cell activation.
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http://dx.doi.org/10.4049/jimmunol.2100548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8530958PMC
November 2021

Activated PI3Kδ signals compromise plasma cell survival via limiting autophagy and increasing ER stress.

J Exp Med 2021 12 29;218(12). Epub 2021 Sep 29.

Center for Immunity and Immunotherapy, Seattle Children's Research Institute, Seattle, WA.

While phosphatidylinositide 3-kinase delta (PI3Kδ) plays a critical role in humoral immunity, the requirement for PI3Kδ signaling in plasma cells remains poorly understood. Here, we used a conditional mouse model of activated PI3Kδ syndrome (APDS), to interrogate the function of PI3Kδ in plasma cell biology. Mice expressing a PIK3CD gain-of-function mutation (aPIK3CD) in B cells generated increased numbers of memory B cells and mounted an enhanced secondary response but exhibited a rapid decay of antibody levels over time. Consistent with these findings, aPIK3CD expression markedly impaired plasma cell generation, and expression of aPIK3CD intrinsically in plasma cells was sufficient to diminish humoral responses. Mechanistically, aPIK3CD disrupted ER proteostasis and autophagy, which led to increased plasma cell death. Notably, this defect was driven primarily by elevated mTORC1 signaling and modulated by treatment with PI3Kδ-specific inhibitors. Our findings establish an essential role for PI3Kδ in plasma cell homeostasis and suggest that modulating PI3Kδ activity may be useful for promoting and/or thwarting specific immune responses.
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http://dx.doi.org/10.1084/jem.20211035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8485856PMC
December 2021

Activated interleukin-7 receptor signaling drives B-cell acute lymphoblastic leukemia in mice.

Leukemia 2022 Jan 30;36(1):42-57. Epub 2021 Jun 30.

Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA, USA.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype of B-ALL often associated with genetic variants that alter cytokine receptor signaling, including mutations in the interleukin-7 receptor (IL7R). To investigate whether IL7R variants are leukemia-initiating, we built mouse models expressing activated Il7r (aIL7R). B-cell intrinsic aIL7R mice developed spontaneous B-ALL, demonstrating sufficiency of Il7r activating mutations in leukemogenesis. Concomitant introduction of a knock-out allele in the associated adapter protein Lnk (encoded by Sh2b3) or a dominant-negative variant of the transcription factor Ikaros (Ikzf1) increased disease penetrance. The resulting murine leukemias displayed monoclonality and recurrent somatic Kras mutations and efficiently engrafted into immunocompetent mice. Phosphoproteomic analyses of aIL7R leukemic cells revealed constitutive Stat5 signaling and B cell receptor (BCR)-like signaling despite the absence of surface pre-BCR. Finally, in vitro treatment of aIL7R leukemic B-cells with Jak, mTOR, or Syk inhibitors blocked growth, confirming that each pathway is active in this mouse model of IL7R-driven B-ALL.
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http://dx.doi.org/10.1038/s41375-021-01326-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8716641PMC
January 2022

TACI haploinsufficiency protects against BAFF-driven humoral autoimmunity in mice.

Eur J Immunol 2021 09 27;51(9):2225-2236. Epub 2021 Jun 27.

Seattle Children's Research Institute, Seattle, WA, USA.

Polymorphisms in TACI, a BAFF family cytokine receptor, are linked to diverse human immune disorders including common variable immunodeficiency (CVID) and systemic lupus erythematosus (SLE). Functional studies of individual variants show modest impacts on surface TACI expression and/or downstream signal transduction, indicating that relatively subtle variation in TACI activity can impact human B-cell biology. However, significant complexity underlies TACI biology, including both positive and negative regulation of physiologic and pathogenic B-cell responses. To model these contradictory events, we compared the functional impact of TACI deletion on separate models of murine SLE driven by T cell-independent and -dependent breaks in B-cell tolerance. First, we studied whether reduced surface TACI expression was sufficient to protect against progressive BAFF-mediated systemic autoimmunity. Strikingly, despite a relatively modest impact on surface TACI levels, TACI haploinsufficiency markedly reduced pathogenic RNA-associated autoantibody titers and conferred long-term protection from BAFF-driven lupus nephritis. In contrast, B cell-intrinsic TACI deletion exerted a limited impact of autoantibody generation in murine lupus characterized by spontaneous germinal center formation and T cell-dependent humoral autoimmunity. Together, these combined data provide new insights into TACI biology and highlight how TACI signals must be tightly regulated during protective and pathogenic B-cell responses.
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http://dx.doi.org/10.1002/eji.202149244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8410679PMC
September 2021

Germline SAMD9L truncation variants trigger global translational repression.

J Exp Med 2021 05;218(5)

Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA.

SAMD9L is an interferon-induced tumor suppressor implicated in a spectrum of multisystem disorders, including risk for myeloid malignancies and immune deficiency. We identified a heterozygous de novo frameshift variant in SAMD9L in an infant with B cell aplasia and clinical autoinflammatory features who died from respiratory failure with chronic rhinovirus infection. Autopsy demonstrated absent bone marrow and peripheral B cells as well as selective loss of Langerhans and Purkinje cells. The frameshift variant led to expression of a truncated protein with interferon treatment. This protein exhibited a gain-of-function phenotype, resulting in interference in global protein synthesis via inhibition of translational elongation. Using a mutational scan, we identified a region within SAMD9L where stop-gain variants trigger a similar translational arrest. SAMD9L variants that globally suppress translation had no effect or increased mRNA transcription. The complex-reported phenotype likely reflects lineage-dominant sensitivities to this translation block. Taken together, our findings indicate that interferon-triggered SAMD9L gain-of-function variants globally suppress translation.
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http://dx.doi.org/10.1084/jem.20201195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7970252PMC
May 2021

Effective, safe, and sustained correction of murine XLA using a UCOE-BTK promoter-based lentiviral vector.

Mol Ther Methods Clin Dev 2021 Mar 20;20:635-651. Epub 2021 Jan 20.

Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101, USA.

X-linked agammaglobulinemia (XLA) is an immune disorder caused by mutations in Bruton's tyrosine kinase (). BTK is expressed in B and myeloid cells, and its deficiency results in a lack of mature B cells and protective antibodies. We previously reported a lentivirus (LV) BTK replacement therapy that restored B cell development and function in and double knockout mice (a phenocopy of human XLA). In this study, with the goal of optimizing both the level and lineage specificity of BTK expression, we generated LV incorporating the proximal human promoter. Hematopoietic stem cells from mice transduced with this vector rescued lineage-specific expression and restored B cell function in recipients. Next, we tested addition of candidate enhancers and/or ubiquitous chromatin opening elements (UCOEs), as well as codon optimization to improve BTK expression. An Eμ enhancer improved B cell rescue, but increased immunoglobulin G (IgG) autoantibodies. Addition of the UCOE avoided autoantibody generation while improving B cell development and function and reducing vector silencing. An optimized vector containing a truncated UCOE upstream of the BTK promoter and codon-optimized BTK cDNA resulted in stable, lineage-regulated BTK expression that mirrored endogenous BTK, making it a strong candidate for XLA therapy.
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http://dx.doi.org/10.1016/j.omtm.2021.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7907679PMC
March 2021

Multimeric antibodies from antigen-specific human IgM+ memory B cells restrict Plasmodium parasites.

J Exp Med 2021 04;218(4)

Department of Immunology, University of Washington School of Medicine, Seattle, WA.

Multimeric immunoglobulin-like molecules arose early in vertebrate evolution, yet the unique contributions of multimeric IgM antibodies to infection control are not well understood. This is partially due to the difficulty of distinguishing low-affinity IgM, secreted rapidly by plasmablasts, from high-affinity antibodies derived from later-arising memory cells. We developed a pipeline to express B cell receptors (BCRs) from Plasmodium falciparum-specific IgM+ and IgG+ human memory B cells (MBCs) as both IgM and IgG molecules. BCRs from both subsets were somatically hypermutated and exhibited comparable monomeric affinity. Crystallization of one IgM+ MBC-derived antibody complexed with antigen defined a linear epitope within a conserved Plasmodium protein. In its physiological multimeric state, this antibody displayed exponentially higher antigen binding than a clonally identical IgG monomer, and more effectively inhibited P. falciparum invasion. Forced multimerization of this IgG significantly improved both antigen binding and parasite restriction, underscoring how avidity can alter antibody function. This work demonstrates the potential of high-avidity IgM in both therapeutics and vaccines.
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http://dx.doi.org/10.1084/jem.20200942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7938364PMC
April 2021

T cells selectively filter oscillatory signals on the minutes timescale.

Proc Natl Acad Sci U S A 2021 03;118(9)

HHMI, University of California, San Francisco, CA 94158;

T cells experience complex temporal patterns of stimulus via receptor-ligand-binding interactions with surrounding cells. From these temporal patterns, T cells are able to pick out antigenic signals while establishing self-tolerance. Although features such as duration of antigen binding have been examined, our understanding of how T cells interpret signals with different frequencies or temporal stimulation patterns is relatively unexplored. We engineered T cells to respond to light as a stimulus by building an optogenetically controlled chimeric antigen receptor (optoCAR). We discovered that T cells respond to minute-scale oscillations of activation signal by stimulating optoCAR T cells with tunable pulse trains of light. Systematically scanning signal oscillation period from 1 to 150 min revealed that expression of CD69, a T cell activation marker, reached a local minimum at a period of ∼25 min (corresponding to 5 to 15 min pulse widths). A combination of inhibitors and genetic knockouts suggest that this frequency filtering mechanism lies downstream of the Erk signaling branch of the T cell response network and may involve a negative feedback loop that diminishes Erk activity. The timescale of CD69 filtering corresponds with the duration of T cell encounters with self-peptide-presenting APCs observed via intravital imaging in mice, indicating a potential functional role for temporal filtering in vivo. This study illustrates that the T cell signaling machinery is tuned to temporally filter and interpret time-variant input signals in discriminatory ways.
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http://dx.doi.org/10.1073/pnas.2019285118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7936380PMC
March 2021

Functional SARS-CoV-2-Specific Immune Memory Persists after Mild COVID-19.

Cell 2021 01 23;184(1):169-183.e17. Epub 2020 Nov 23.

Department of Immunology, University of Washington School of Medicine, Seattle, WA 98109, USA. Electronic address:

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is causing a global pandemic, and cases continue to rise. Most infected individuals experience mildly symptomatic coronavirus disease 2019 (COVID-19), but it is unknown whether this can induce persistent immune memory that could contribute to immunity. We performed a longitudinal assessment of individuals recovered from mild COVID-19 to determine whether they develop and sustain multifaceted SARS-CoV-2-specific immunological memory. Recovered individuals developed SARS-CoV-2-specific immunoglobulin (IgG) antibodies, neutralizing plasma, and memory B and memory T cells that persisted for at least 3 months. Our data further reveal that SARS-CoV-2-specific IgG memory B cells increased over time. Additionally, SARS-CoV-2-specific memory lymphocytes exhibited characteristics associated with potent antiviral function: memory T cells secreted cytokines and expanded upon antigen re-encounter, whereas memory B cells expressed receptors capable of neutralizing virus when expressed as monoclonal antibodies. Therefore, mild COVID-19 elicits memory lymphocytes that persist and display functional hallmarks of antiviral immunity.
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http://dx.doi.org/10.1016/j.cell.2020.11.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682481PMC
January 2021

Multiplexed Functional Assessment of Genetic Variants in CARD11.

Am J Hum Genet 2020 12 16;107(6):1029-1043. Epub 2020 Nov 16.

Seattle Children's Research Institute, Seattle, WA 98101, USA; Department of Pediatrics, University of Washington, Seattle, WA 98195, USA; Department of Pharmacology, University of Washington, Seattle, WA 98195, USA; Brotman-Baty Institute for Precision Medicine, Seattle, WA 98195, USA. Electronic address:

Genetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a "cloning-free" saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.
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http://dx.doi.org/10.1016/j.ajhg.2020.10.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820631PMC
December 2020

Generation of a cost-effective cell line for support of high-throughput isolation of primary human B cells and monoclonal neutralizing antibodies.

J Immunol Methods 2021 01 15;488:112901. Epub 2020 Oct 15.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Department of Global Health, University of Washington, Seattle, WA, USA; Deparment of Laboratory Medicine and Pahthology, University of Washington, Seattle, WA, USA. Electronic address:

The isolation of human monoclonal antibodies (mAbs) arising from natural infection with human pathogens has proven to be a powerful technology, facilitating the understanding of the host response to infection at a molecular level. mAbs can reveal sites of vulnerability on pathogens and illuminate the biological function of the antigenic targets. Moreover, mAbs have the potential to be used directly for therapeutic applications such as passive delivery to prevent infection in susceptible target populations, and as treatment of established infection. The isolation of antigen-specific B cells from vaccine trials can also assist in deciphering whether the desired B cells are being targeted by a given vaccine. Several different processes have been developed to isolate mAbs, but all are generally labor-intensive and result in varying degrees of efficiency. Here, we describe the development of a cost-effective feeder cell line that stably expresses CD40-ligand, interleukin-2 and interleukin-21. Sorting of single B cells onto a layer of irradiated feeder cells sustained antibody production that permits functional screening of secreted antibodies in a manner that enables subsequent recovery of B cells for recombinant antibody cloning. As a proof of concept, we show that this approach can be used to isolate B cells that secrete antibodies that neutralize human papilloma virus (HPV) from participants of an HPV vaccine study.
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http://dx.doi.org/10.1016/j.jim.2020.112901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7560121PMC
January 2021

Functional SARS-CoV-2-specific immune memory persists after mild COVID-19.

Res Sq 2020 Aug 13. Epub 2020 Aug 13.

Department of Immunology, University of Washington School of Medicine, Seattle, WA, USA.

The recently emerged SARS-CoV-2 virus is currently causing a global pandemic and cases continue to rise. The majority of infected individuals experience mildly symptomatic coronavirus disease 2019 (COVID-19), but it is unknown whether this can induce persistent immune memory that might contribute to herd immunity. Thus, we performed a longitudinal assessment of individuals recovered from mildly symptomatic COVID-19 to determine if they develop and sustain immunological memory against the virus. We found that recovered individuals developed SARS-CoV-2-specific IgG antibody and neutralizing plasma, as well as virus-specific memory B and T cells that not only persisted, but in some cases increased numerically over three months following symptom onset. Furthermore, the SARS-CoV-2-specific memory lymphocytes exhibited characteristics associated with potent antiviral immunity: memory T cells secreted IFN-γ and expanded upon antigen re-encounter, while memory B cells expressed receptors capable of neutralizing virus when expressed as antibodies. These findings demonstrate that mild COVID-19 elicits memory lymphocytes that persist and display functional hallmarks associated with antiviral protective immunity.
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http://dx.doi.org/10.21203/rs.3.rs-57112/v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430600PMC
August 2020

Functional SARS-CoV-2-specific immune memory persists after mild COVID-19.

medRxiv 2020 Aug 15. Epub 2020 Aug 15.

The recently emerged SARS-CoV-2 virus is currently causing a global pandemic and cases continue to rise. The majority of infected individuals experience mildly symptomatic coronavirus disease 2019 (COVID-19), but it is unknown whether this can induce persistent immune memory that might contribute to herd immunity. Thus, we performed a longitudinal assessment of individuals recovered from mildly symptomatic COVID-19 to determine if they develop and sustain immunological memory against the virus. We found that recovered individuals developed SARS-CoV-2-specific IgG antibody and neutralizing plasma, as well as virus-specific memory B and T cells that not only persisted, but in some cases increased numerically over three months following symptom onset. Furthermore, the SARS-CoV-2-specific memory lymphocytes exhibited characteristics associated with potent antiviral immunity: memory T cells secreted IFN-γ and expanded upon antigen re-encounter, while memory B cells expressed receptors capable of neutralizing virus when expressed as antibodies. These findings demonstrate that mild COVID-19 elicits memory lymphocytes that persist and display functional hallmarks associated with antiviral protective immunity.
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http://dx.doi.org/10.1101/2020.08.11.20171843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430599PMC
August 2020

Neutralizing Antibodies Protect against Oral Transmission of Lymphocryptovirus.

Cell Rep Med 2020 Jun;1(3)

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

Epstein-Barr virus (EBV) is a cancer-associated pathogen for which there is no vaccine. Successful anti-viral vaccines elicit antibodies that neutralize infectivity; however, it is unknown whether neutralizing antibodies prevent EBV acquisition. Here we assessed whether passively delivered AMMO1, a monoclonal antibody that neutralizes EBV in a cell-type-independent manner, could protect against experimental EBV challenge in two animal infection models. When present prior to a high-dose intravenous EBV challenge, AMMO1 prevented viremia and reduced viral loads to nearly undetectable levels in humanized mice. AMMO1 conferred sterilizing immunity to three of four macaques challenged orally with rhesus lymphocryptovirus, the EBV ortholog that infects rhesus macaques. The infected macaque had lower plasma neutralizing activity than the protected animals. These results indicate that a vaccine capable of eliciting adequate titers of neutralizing antibodies targeting the AMMO1 epitope may protect against EBV acquisition and are therefore highly relevant to the design of an effective EBV vaccine.
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http://dx.doi.org/10.1016/j.xcrm.2020.100033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7386402PMC
June 2020

Gene editing to induce FOXP3 expression in human CD4 T cells leads to a stable regulatory phenotype and function.

Sci Transl Med 2020 06;12(546)

Center for Immunity and Immunotherapies and the Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.

Thymic regulatory T cells (tT) are potent inhibitors of autoreactive immune responses, and loss of tT function results in fatal autoimmune disease. Defects in tT number or function are also implicated in multiple autoimmune diseases, leading to growing interest in use of T as cell therapies to establish immune tolerance. Because tT are present at low numbers in circulating blood and may be challenging to purify and expand and also inherently defective in some subjects, we designed an alternative strategy to create autologous T-like cells from bulk CD4 T cells. We used homology-directed repair (HDR)-based gene editing to enforce expression of FOXP3, the master transcription factor for tT Targeted insertion of a robust enhancer/promoter proximal to the first coding exon bypassed epigenetic silencing, permitting stable and robust expression of endogenous FOXP3. HDR-edited T cells, edT, manifested a transcriptional program leading to sustained expression of canonical markers and suppressive activity of tT Both human and murine edT mediated immunosuppression in vivo in models of inflammatory disease. Further, this engineering strategy permitted generation of antigen-specific edT with robust in vitro and in vivo functional activity. Last, edT could be enriched and expanded at scale using clinically relevant methods. Together, these findings suggest that edT production may permit broad future clinical application.
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http://dx.doi.org/10.1126/scitranslmed.aay6422DOI Listing
June 2020

Optimizing lentiviral vector transduction of hematopoietic stem cells for gene therapy.

Gene Ther 2020 12 27;27(12):545-556. Epub 2020 Apr 27.

Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.

Autologous gene therapy using lentiviral vectors (LVs) holds promise for treating monogenetic blood diseases. However, clinical applications can be limited by suboptimal hematopoietic stem cell (HSC) transduction and insufficient quantities of available vector. We recently reported gene therapy for X-linked severe combined immunodeficiency using a protocol in which patient CD34 cells were incubated with two successive transductions. Here we describe an improved protocol for LV delivery to CD34 cells that simplifies product manipulation, reduces vector consumption, and achieves greater vector copy number (VCN) of repopulating HSCs in mouse xenotransplantation assays. Notable findings include the following: (1) the VCN of CD34 cells measured shortly after transduction did not always correlate with the VCN of repopulating HSCs after xenotransplantation; (2) single-step transduction at higher CD34 cell concentrations (2-4 × 10/ml) conserved LV without compromising HSC VCN; (3) poloxamer F108 (LentiBOOST) increased HSC VCN by two- to threefold (average from three donors); (4) although LentiBOOST + prostaglandin E2 combination further increased VCN in vitro, the VCN observed in vivo were similar to LentiBOOST alone; (5) cyclosporine H increased the HSC VCN to a similar or greater extent with LentiBOOST in vivo. Our findings delineate an improved protocol to increase the VCN of HSCs after CD34 cell transduction with clinically relevant LVs.
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http://dx.doi.org/10.1038/s41434-020-0150-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606410PMC
December 2020

Excellent outcomes following hematopoietic cell transplantation for Wiskott-Aldrich syndrome: a PIDTC report.

Blood 2020 06;135(23):2094-2105

Division of Pediatric Hematology/Oncology, Primary Children's Hospital, University of Utah School of Medicine, Salt Lake City, UT.

Wiskott-Aldrich syndrome (WAS) is an X-linked disease caused by mutations in the WAS gene, leading to thrombocytopenia, eczema, recurrent infections, autoimmune disease, and malignancy. Hematopoietic cell transplantation (HCT) is the primary curative approach, with the goal of correcting the underlying immunodeficiency and thrombocytopenia. HCT outcomes have improved over time, particularly for patients with HLA-matched sibling and unrelated donors. We report the outcomes of 129 patients with WAS who underwent HCT at 29 Primary Immune Deficiency Treatment Consortium centers from 2005 through 2015. Median age at HCT was 1.2 years. Most patients (65%) received myeloablative busulfan-based conditioning. With a median follow-up of 4.5 years, the 5-year overall survival (OS) was 91%. Superior 5-year OS was observed in patients <5 vs ≥5 years of age at the time of HCT (94% vs 66%; overall P = .0008). OS was excellent regardless of donor type, even in cord blood recipients (90%). Conditioning intensity did not affect OS, but was associated with donor T-cell and myeloid engraftment after HCT. Specifically, patients who received fludarabine/melphalan-based reduced-intensity regimens were more likely to have donor myeloid chimerism <50% early after HCT. In addition, higher platelet counts were observed among recipients who achieved full (>95%) vs low-level (5%-49%) donor myeloid engraftment. In summary, HCT outcomes for WAS have improved since 2005, compared with prior reports. HCT at a younger age continues to be associated with superior outcomes supporting the recommendation for early HCT. High-level donor myeloid engraftment is important for platelet reconstitution after either myeloablative or busulfan-containing reduced intensity conditioning. (This trial was registered at www.clinicaltrials.gov as #NCT02064933.).
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http://dx.doi.org/10.1182/blood.2019002939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7273831PMC
June 2020

Phosphatase PTPN22 Regulates Dendritic Cell Homeostasis and cDC2 Dependent T Cell Responses.

Front Immunol 2020 4;11:376. Epub 2020 Mar 4.

Faculty of Life Sciences and Medicine, Centre for Inflammation Biology and Cancer Immunology, King's College London, London, United Kingdom.

Dendritic cells (DCs) are specialized antigen presenting cells that instruct T cell responses through sensing environmental and inflammatory danger signals. Maintaining the homeostasis of the multiple functionally distinct conventional dendritic cells (cDC) subsets that exist is crucial for regulating immune responses, with changes in numbers sufficient to break immune tolerance. Using mice we demonstrate that the phosphatase PTPN22 is a highly selective, negative regulator of cDC2 homeostasis, preventing excessive population expansion from as early as 3 weeks of age. Mechanistically, PTPN22 mediates cDC2 homeostasis in a cell intrinsic manner by restricting cDC2 proliferation. A single nucleotide polymorphism, PTPN22, is one of the strongest genetic risk factors for multiple autoantibody associated human autoimmune diseases. We demonstrate that cDC2 are also expanded in mice carrying the orthologous PTPN22 mutation. As a consequence, cDC2 dependent CD4 T cell proliferation and T follicular helper cell responses are increased. Collectively, our data demonstrate that PTPN22 controls cDC2 homeostasis, which in turn ensures appropriate cDC2-dependent T cell responses under antigenic challenge. Our findings provide a link between perturbations in DC development and susceptibility to a broad spectrum of PTPN22 associated human autoimmune diseases.
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http://dx.doi.org/10.3389/fimmu.2020.00376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065600PMC
March 2021

Individual and combined effects of the infrared, visible, and ultraviolet light components of solar radiation on damage biomarkers in human skin cells.

FASEB J 2020 03 16;34(3):3874-3883. Epub 2020 Jan 16.

Dermatological Sciences, Translational and Clinical Research Institute, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.

The ability of solar ultraviolet (UV) to induce skin cancer and photoaging is well recognized. The effect of the infrared (IR) and visible light (Vis) components of solar radiation on skin and their interaction with UV is less well known. This study compared the effects of physiologically relevant doses of complete (UV + Vis + IR) solar-simulated light and its individual components on matched primary dermal fibroblasts and epidermal keratinocytes from human donors on three biomarkers of cellular damage (reactive oxygen species (ROS) generation, mitochondrial DNA (mtDNA), and nuclear DNA (nDNA) damage). There was a greater induction of ROS, mtDNA, and nDNA damage with the inclusion of the visible and IR components of solar-simulated light in primary fibroblast cells compared to primary keratinocytes (P < .001). Experiments using exposure to specific components of solar light alone or in combination showed that the UV, Vis, and IR components of solar light synergistically increased ROS generation in primary fibroblasts but not primary keratinocytes (P < .001). Skin cell lines were used to confirm these findings. These observations have important implications for different skin cell type responses to the individual and interacting components of solar light and therefore photodamage mechanisms and photoprotection interventions.
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http://dx.doi.org/10.1096/fj.201902351RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079185PMC
March 2020

Efficient CRISPR/Cas9 Disruption of Autoimmune-Associated Genes Reveals Key Signaling Programs in Primary Human T Cells.

J Immunol 2019 12 13;203(12):3166-3178. Epub 2019 Nov 13.

Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101;

Risk of autoimmunity is associated with multiple genetic variants. Genome-wide association studies have linked single-nucleotide polymorphisms in the phosphatases (rs2476601) and (rs1893217) to increased risk for multiple autoimmune diseases. Previous mouse studies of loss of function or risk variants in these genes revealed hyperactive T cell responses, whereas studies of human lymphocytes revealed contrasting phenotypes. To better understand this dichotomy, we established a robust gene editing platform to rapidly address the consequences of loss of function of candidate genes in primary human CD4 T cells. Using CRISPR/Cas9, we obtained efficient gene disruption (>80%) of target genes encoding proteins involved in Ag and cytokine receptor signaling pathways including and Loss-of-function data in all genes studied correlated with previous data from mouse models. Further analyses of gene-disrupted T cells demonstrated dynamic effects, by which hyperactive IL-2R signaling promoted compensatory transcriptional events, eventually resulting in T cells that were hyporesponsive to IL-2. These results imply that altered phosphatase activity promotes evolving phenotypes based on Ag experience and/or other programming signals. This approach enables the discovery of molecular mechanisms modulating risk of autoimmunity that have been difficult to parse in traditional mouse models or cross-sectional human studies.
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http://dx.doi.org/10.4049/jimmunol.1900848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904544PMC
December 2019

Functional Characterization of CD11c Age-Associated B Cells as Memory B Cells.

J Immunol 2019 12 21;203(11):2817-2826. Epub 2019 Oct 21.

Seattle Children's Research Institute, Seattle, WA 98101;

Age-associated B cells (ABCs) are a unique subset of B cells defined by surface CD11b and CD11c expression. Although ABC expansion has been observed in both human and animal studies in the setting of advanced age, during humoral autoimmunity and following viral infection, the functional properties of this cellular subset remain incompletely defined. In the current study, we demonstrate that ABCs fulfill the criteria for memory B cells (MBCs), based on evidence of Ag-dependent expansion and persistence in a state poised for rapid differentiation into Ab-secreting plasma cells during secondary responses. First, we show that a majority of ABCs are not actively cycling but exhibit an extensive replication history consistent with prior Ag engagement. Second, despite unswitched surface IgM expression, ABCs show evidence of activation-induced cytidine deaminase (AID)-dependent somatic hypermutation. Third, BCRs cloned from sorted ABCs exhibit broad autoreactivity and polyreactivity. Although the overall level of ABC self-reactivity was not increased relative to naive B cells, ABCs lacked features of functional anergy characteristic of autoreactive B cells. Fourth, ABCs express MBC surface markers consistent with being poised for rapid plasma cell differentiation during recall responses. Finally, in a murine model of viral infection, adoptively transferred CD11c B cells rapidly differentiated into class-switched Ab-secreting cells upon Ag rechallenge. In summary, we phenotypically and functionally characterize ABCs as IgM-expressing MBCs, findings that together implicate ABCs in the pathogenesis of systemic autoimmunity.
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http://dx.doi.org/10.4049/jimmunol.1900404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6864310PMC
December 2019

Loss of PTPN22 abrogates the beneficial effect of cohousing-mediated fecal microbiota transfer in murine colitis.

Mucosal Immunol 2019 11 9;12(6):1336-1347. Epub 2019 Sep 9.

Department of Gastroenterology and Hepatology, University Hospital and University of Zurich, Zurich, Switzerland.

Fecal microbiota transfer (FMT) is a very efficient approach for the treatment of severe and recurring C. difficile infections. However, the beneficial effect of FMT in other disorders such as ulcerative colitis (UC) or Crohn's disease remains unclear. Furthermore, it is currently unknown how disease-associated genetic variants in donors or recipients influence the effect of FMT. We found that bacteria-transfer from wild-type (WT) donors via cohousing was efficient in inducing recovery from colitis in WT mice, but not in mice deficient in protein-tyrosine phosphatase non-receptor type 22 (PTPN22), a known risk gene for several chronic inflammatory diseases. Also cohousing of PTPN22-deficient mice with diseased WT mice failed to induce faster recovery. Our data indicate that the genetic background of the donor and the recipient influences the outcome of microbiota transfer, and offers a potential explanation why transfer of fecal microbes from some, but not all donors is efficient in UC patients.
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http://dx.doi.org/10.1038/s41385-019-0201-1DOI Listing
November 2019

Therapeutically relevant engraftment of a CRISPR-Cas9-edited HSC-enriched population with HbF reactivation in nonhuman primates.

Sci Transl Med 2019 07;11(503)

Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

Reactivation of fetal hemoglobin (HbF) is being pursued as a treatment strategy for hemoglobinopathies. Here, we evaluated the therapeutic potential of hematopoietic stem and progenitor cells (HSPCs) edited with the CRISPR-Cas9 nuclease platform to recapitulate naturally occurring mutations identified in individuals who express increased amounts of HbF, a condition known as hereditary persistence of HbF. CRISPR-Cas9 treatment and transplantation of HSPCs purified on the basis of surface expression of the CD34 receptor in a nonhuman primate (NHP) autologous transplantation model resulted in up to 30% engraftment of gene-edited cells for >1 year. Edited cells effectively and stably reactivated HbF, as evidenced by up to 18% HbF-expressing erythrocytes in peripheral blood. Similar results were obtained by editing highly enriched stem cells, defined by the markers CD34CD90CD45RA, allowing for a 10-fold reduction in the number of transplanted target cells, thus considerably reducing the need for editing reagents. The frequency of engrafted, gene-edited cells persisting in vivo using this approach may be sufficient to ameliorate the phenotype for a number of genetic diseases.
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http://dx.doi.org/10.1126/scitranslmed.aaw3768DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8407476PMC
July 2019

In Vivo Outcome of Homology-Directed Repair at the HBB Gene in HSC Using Alternative Donor Template Delivery Methods.

Mol Ther Nucleic Acids 2019 Sep 7;17:277-288. Epub 2019 Jun 7.

Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA, USA; Department of Pediatrics, University of Washington, School of Medicine, Seattle, WA, USA; Department of Immunology, University of Washington, School of Medicine, Seattle, WA, USA. Electronic address:

Gene editing following designer nuclease cleavage in the presence of a DNA donor template can revert mutations in disease-causing genes. For optimal benefit, reversion of the point mutation in HBB leading to sickle cell disease (SCD) would permit precise homology-directed repair (HDR) while concurrently limiting on-target non-homologous end joining (NHEJ)-based HBB disruption. In this study, we directly compared the relative efficiency of co-delivery of a novel CRISPR/Cas9 ribonucleoprotein targeting HBB in association with recombinant adeno-associated virus 6 (rAAV6) versus single-stranded oligodeoxynucleotides (ssODNs) to introduce the sickle mutation (GTC or GTG; encoding E6V) or a silent change (GAA; encoding E6optE) in human CD34 mobilized peripheral blood stem cells (mPBSCs) derived from healthy donors. In vitro, rAAV6 outperformed ssODN donor template delivery and mediated greater HDR correction, leading to both higher HDR rates and a higher HDR:NHEJ ratio. In contrast, at 12-14 weeks post-transplant into recipient, immunodeficient, NOD, B6, SCID Il2rγ Kit(W41/W41) (NBSGW) mice, a ∼6-fold higher proportion of ssODN-modified cells persisted in vivo compared to recipients of rAAV6-modified mPBSCs. Together, our findings highlight that methodology for donor template delivery markedly impacts long-term persistence of HBB gene-modified mPBSCs, and they suggest that the ssODN platform is likely to be most amenable to direct clinical translation.
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http://dx.doi.org/10.1016/j.omtn.2019.05.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611979PMC
September 2019

Protein tyrosine phosphatase non-receptor type 22 modulates colitis in a microbiota-dependent manner.

J Clin Invest 2019 05 20;129(6):2527-2541. Epub 2019 May 20.

Department of Gastroenterology and Hepatology, University Hospital Zurich and University of Zurich, Zurich, Switzerland.

The gut microbiota is crucial for our health, and well-balanced interactions between the host's immune system and the microbiota are essential to prevent chronic intestinal inflammation, as observed in inflammatory bowel diseases (IBD). A variant in protein tyrosine phosphatase non-receptor type 22 (PTPN22) is associated with reduced risk of developing IBD, but promotes the onset of autoimmune disorders. While the role of PTPN22 in modulating molecular pathways involved in IBD pathogenesis is well studied, its impact on shaping the intestinal microbiota has not been addressed in depth. Here, we demonstrate that mice carrying the PTPN22 variant (619W mice) were protected from acute dextran sulfate sodium (DSS) colitis, but suffered from pronounced inflammation upon chronic DSS treatment. The basal microbiota composition was distinct between genotypes, and DSS-induced dysbiosis was milder in 619W mice than in WT littermates. Transfer of microbiota from 619W mice after the first DSS cycle into treatment-naive 619W mice promoted colitis, indicating that changes in microbial composition enhanced chronic colitis in those animals. This indicates that presence of the PTPN22 variant affects intestinal inflammation by modulating the host's response to the intestinal microbiota.
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http://dx.doi.org/10.1172/JCI123263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546451PMC
May 2019

Dynamic Immune Phenotypes of B and T Helper Cells Mark Distinct Stages of T1D Progression.

Diabetes 2019 06 20;68(6):1240-1250. Epub 2019 Mar 20.

Translational Research Program, Benaroya Research Institute, Seattle, WA

Multiple studies of B- and T-cell compartments and their response to stimuli demonstrate alterations in established type 1 diabetes (T1D). Yet it is not known whether these alterations reflect immune mechanisms that initiate islet autoimmunity, promote disease progression, or are secondary to disease. To address these questions, we used samples from the TrialNet Pathway to Prevention study to investigate T-cell responses to interleukin (IL)-2 and regulatory T cell-mediated suppression, the composition of the B-cell compartment, and B-cell responses to B-cell receptor and IL-21 receptor engagement. These studies revealed stage-dependent T- and B-cell functional and immune phenotypes; namely, early features that differentiate autoantibody-positive at-risk first-degree relatives (FDRs) from autoantibody-negative FDRs and persisted through clinical diagnosis; late features that arose at or near T1D diagnosis; and dynamic features that were enhanced early and blunted at later disease stages, indicating evolving responses along the continuum of T1D. We further explored how these specific phenotypes are influenced by therapeutic interventions. Our integrated studies provide unique insights into stable and dynamic stage-specific immune states and define novel immune phenotypes of potential clinical relevance.
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http://dx.doi.org/10.2337/db18-1081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610015PMC
June 2019

The -P1104A Autoimmune Protective Variant Limits Coordinate Signals Required to Generate Specialized T Cell Subsets.

Front Immunol 2019 25;10:44. Epub 2019 Jan 25.

Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA, United States.

TYK2 is a JAK family member that functions downstream of multiple cytokine receptors. Genome wide association studies have linked a SNP (rs34536443) within encoding a Proline to Alanine substitution at amino acid 1104, to protection from multiple autoimmune diseases including systemic lupus erythematosus (SLE) and multiple sclerosis (MS). The protective role of this SNP in autoimmune pathogenesis, however, remains incompletely understood. Here we found that T follicular helper (Tfh) cells, switched memory B cells, and IFNAR signaling were decreased in healthy individuals that expressed the protective variant ( ). To study this variant , we developed a knock-in murine model of this allele. Murine expressing T cells homozygous for the protective allele, but not cells heterozygous for this change, manifest decreased IL-12 receptor signaling, important for Tfh lineage commitment. Further, homozygous T cells exhibited diminished Th1 skewing. Surprisingly, despite these signaling changes, formation of Tfh and GC B cells was unaffected in two models of T cell dependent immune responses and in two alternative SLE models. TYK2 is also activated downstream of IL-23 receptor engagement. Here, we found that expressing T cells had reduced IL-23 dependent signaling as well as a diminished ability to skew toward Th17 . Consistent with these findings, homozygous, but not heterozygous, mice were fully protected in a murine model of MS. Homozygous mice had fewer infiltrating CD4 T cells within the CNS. Most strikingly, homozygous mice had a decreased proportion of IL-17/IFNγ, double positive, pathogenic CD4 T cells in both the draining lymph nodes (LN) and CNS. Thus, in an autoimmune model, such as EAE, impacted by both altered Th1 and Th17 signaling, the allele can effectively shield animals from disease. Taken together, our findings suggest that TYK2 diminishes IL-12, IL-23, and IFN I signaling and that its protective effect is most likely manifest in the setting of autoimmune triggers that concurrently dysregulate at least two of these important signaling cascades.
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http://dx.doi.org/10.3389/fimmu.2019.00044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355696PMC
December 2019

TALEN-Mediated Gene Editing of in Human Hematopoietic Stem Cells Leads to Therapeutic Fetal Hemoglobin Induction.

Mol Ther Methods Clin Dev 2019 Mar 31;12:175-183. Epub 2018 Dec 31.

Center for Immunity and Immunotherapies and the Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.

Elements within the γ-hemoglobin promoters ( and ) function to bind transcription complexes that mediate repression of fetal hemoglobin expression. Sickle cell disease (SCD) subjects with a 13-bp deletion in the promoter exhibit a clinically favorable hereditary persistence of fetal hemoglobin (HPFH) phenotype. We developed TALENs targeting the homologous promoters to de-repress fetal hemoglobin. Transfection of human CD34 cells with TALEN mRNA resulted in indel generation in (43%) and (74%) including the 13-bp HPFH deletion (∼6%). Erythroid differentiation of edited cells revealed a 4.6-fold increase in γ-hemoglobin expression as detected by HPLC. Assessment of TALEN-edited CD34 cells in a humanized mouse model demonstrated sustained presence of indels in hematopoietic cells up to 24 weeks. Indel rates remained unchanged following secondary transplantation consistent with editing of long-term repopulating stem cells (LT-HSCs). Human γ-hemoglobin expressing F cells were detected by flow cytometry approximately 50% more frequently in edited animals compared to mock. Together, these findings demonstrate that TALEN-mediated indel generation in the γ-hemoglobin promoter leads to high levels of fetal hemoglobin expression and , suggesting that this approach can provide therapeutic benefit in patients with SCD or β-thalassemia.
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http://dx.doi.org/10.1016/j.omtm.2018.12.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6348980PMC
March 2019
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