Publications by authors named "David J Rawlings"

143 Publications

T cells selectively filter oscillatory signals on the minutes timescale.

Proc Natl Acad Sci U S A 2021 Mar;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
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 Jan 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

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 Dec 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

Pediatric Allergy, Immunology, and Blood and Marrow Transplant Division, University of California, San Francisco Benioff Children's Hospital, San Francisco, CA.

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 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

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
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

The long and the short of it: insights into the cellular source of autoantibodies as revealed by B cell depletion therapy.

Curr Opin Immunol 2018 12 1;55:81-88. Epub 2018 Nov 1.

Seattle Children's Research Institute, Seattle, WA, United States; Department of Pediatrics, University of Washington, School of Medicine, United States. Electronic address:

High titers of pathogenic autoantibodies are a hallmark of many autoimmune diseases. However, much remains unknown about the self-reactive plasma cells that are key mediators of disease. We propose a model in which the varying efficacy of precursor B cell depletion for the treatment of humoral autoimmunity can be explained by differences in the relative contributions of pathogenic antibodies by short-lived versus long-lived plasma cells. Beyond therapeutic considerations, this model suggests that we can infer the cellular source of disease-associated autoantibodies by the durability of serum titers following B cell depletion. Data from clinical trials and animal models across different autoimmune diseases may provide useful insights into the lifespan, lifestyle and fate of autoreactive plasma cells.
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http://dx.doi.org/10.1016/j.coi.2018.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289281PMC
December 2018

Integrated B Cell, Toll-like, and BAFF Receptor Signals Promote Autoantibody Production by Transitional B Cells.

J Immunol 2018 12 29;201(11):3258-3268. Epub 2018 Oct 29.

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

The B cell survival cytokine BAFF has been linked with the pathogenesis of systemic lupus erythematosus (SLE). BAFF binds distinct BAFF-family surface receptors, including the BAFF-R and transmembrane activator and CAML interactor (TACI). Although originally characterized as a negative regulator of B cell activation, TACI signals are critical for class-switched autoantibody (autoAb) production in BAFF transgenic mice. Consistent with this finding, a subset of transitional splenic B cells upregulate surface TACI expression and contribute to BAFF-driven autoAb. In the current study, we interrogated the B cell signals required for transitional B cell TACI expression and Ab production. Surprisingly, despite established roles for dual BCR and TLR signals in autoAb production in SLE, signals downstream of these receptors exerted distinct impacts on transitional B cell TACI expression and autoAb titers. Whereas loss of BCR signals prevented transitional B cell TACI expression and resulted in loss of serum autoAb across all Ig isotypes, lack of TLR signals exerted a more limited impact restricted to autoAb class-switch recombination without altering transitional B cell TACI expression. Finally, in parallel with the protective effect of TACI deletion, loss of BAFF-R signaling also protected against BAFF-driven autoimmunity. Together, these findings highlight how multiple signaling pathways integrate to promote class-switched autoAb production by transitional B cells, events that likely impact the pathogenesis of SLE and other BAFF-dependent autoimmune diseases.
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http://dx.doi.org/10.4049/jimmunol.1800393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779322PMC
December 2018

Generation of functional murine CD11c age-associated B cells in the absence of B cell T-bet expression.

Eur J Immunol 2019 01 5;49(1):170-178. Epub 2018 Nov 5.

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

Age-associated B cells (ABC), a novel subset of activated B cells defined by CD11b and CD11c expression, have been linked with both protective anti-viral responses and the pathogenesis of systemic autoimmunity. Expression of the T 1 lineage transcription factor T-bet has been identified as a defining feature of ABC biology, with B cell-intrinsic expression of this transcription factor proposed to be required for ABC formation. In contrast to this model, we report that Tbx21 (encoding T-bet)-deficient B cells upregulate CD11b and CD11c surface expression in vitro in response to integrated TLR and cytokine signals. Moreover, B cell-intrinsic T-bet deletion in a murine lupus model exerted no impact of ABC generation in vivo, with Tbx21 ABCs exhibiting an identical surface phenotype to wild-type (WT) ABCs. Importantly, WT and Tbx21 ABCs sorted from autoimmune mice produced equivalent amounts of IgM and IgG ex vivo following TLR stimulation, indicating that T-bet-deficient ABCs are likely functional in vivo. In summary, our data contradict the established literature by demonstrating that T-bet expression is not uniformly required for ABC generation.
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http://dx.doi.org/10.1002/eji.201847641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779321PMC
January 2019

Activated PIK3CD drives innate B cell expansion yet limits B cell-intrinsic immune responses.

J Exp Med 2018 10 7;215(10):2485-2496. Epub 2018 Sep 7.

Seattle Children's Research Institute, Seattle, WA

Activated PI3K-delta syndrome (APDS) is an immunodeficiency caused by gain-of-function mutations in PIK3CD. This disease exhibits complex immune phenotypes including increased IgM, recurrent infection, and impaired vaccine responses. To better understand the impact of B cells in this disease, we generated an inducible model of the common APDS mutation (h-E1021K; referred to as aPIK3CD) and intercrossed these mice with B cell-specific Cre models. Mb1-aPIK3CD mice exhibited bone marrow B lymphopenia and, conversely, expansion of the peripheral innate B1a and MZ B cell compartments. aPIK3CD B cells manifest increased pS6 and increased survival at several stages, without alterations in cycling, and baseline increases in plasma cells, natural IgM, and IgG3. Finally, Mb1-aPIK3CD mice exhibited blunted T cell-independent immune responses, and both AID- and CD21-aPIK3CD mice displayed reduced class-switched antibodies following T cell-dependent immunization. Thus, aPIK3CD alters B cell development and function and is counter-productive during immune responses, providing insight into B cell-intrinsic contributions to the APDS phenotype.
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http://dx.doi.org/10.1084/jem.20180617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6170176PMC
October 2018

Activated CARD11 accelerates germinal center kinetics, promoting mTORC1 and terminal differentiation.

J Exp Med 2018 09 20;215(9):2445-2461. Epub 2018 Aug 20.

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

Activating mutations in the adapter protein CARD11 associated with diffuse large B cell lymphomas (DLBCLs) are predicted to arise during germinal center (GC) responses, leading to inappropriate activation of NF-κB signaling. Here, we modeled the B cell-intrinsic impact of the L251P activating mutation in CARD11 (aCARD11) on the GC response. Global B cell aCARD11 expression led to a modest increase in splenic B cells and a severe reduction in B1 B cell numbers, respectively. Following T cell-dependent immunization, aCARD11 cells exhibited increased rates of GC formation, resolution, and differentiation. Restriction of aCARD11 to GC B cells similarly altered the GC response and B cell differentiation. In this model, aCARD11 promoted dark zone skewing along with increased cycling, AID levels, and class switch recombination. Furthermore, aCard11 GC B cells displayed increased biomass and mTORC1 signaling, suggesting a novel strategy for targeting aCARD11-driven DLBCL. While aCARD11 potently impacts GC responses, the rapid GC contraction suggests it requires collaboration with events that limit terminal differentiation to promote lymphoma.
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http://dx.doi.org/10.1084/jem.20180230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6122963PMC
September 2018

Protein tyrosine phosphatase PTPN22 regulates LFA-1 dependent Th1 responses.

J Autoimmun 2018 11 24;94:45-55. Epub 2018 Jul 24.

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

A missense C1858T single nucleotide polymorphism within PTPN22 is a strong genetic risk factor for the development of multiple autoimmune diseases. PTPN22 encodes a protein tyrosine phosphatase that negatively regulates immuno-receptor proximal Src and Syk family kinases. Notably, PTPN22 negatively regulates kinases downstream of T-cell receptor (TCR) and LFA-1, thereby setting thresholds for T-cell activation. Alterations to the quality of TCR and LFA-1 engagement at the immune synapse and the regulation of downstream signals can have profound effects on the type of effector T-cell response induced. Here we describe how IFNγ Th1 responses are potentiated in Ptpn22 T-cells and in T-cells from mice expressing Ptpn22 (the mouse orthologue of the human genetic variant) as they age, or following repeated immune challenge, and explore the mechanisms contributing to the expansion of Th1 cells. Specifically, we uncover two LFA-1-ICAM dependent mechanisms; one T-cell intrinsic, and one T-cell extrinsic. Firstly, we found that in vitro anti-CD3/LFA-1 induced Th1 responses were enhanced in Ptpn22 T-cells compared to WT, whereas anti-CD3/anti-CD28 induced IFNy responses were similar. These data were associated with an enhanced ability of Ptpn22 T-cells to engage ICAM-1 at the immune synapse when incubated on planar lipid bilayers, and to form conjugates with dendritic cells. Secondly, we observed a T-cell extrinsic mechanism whereby repeated stimulation of WT OT-II T-cells with LPS and OVA pulsed Ptpn22 bone marrow derived dendritic cells (BMDCs) was sufficient to enhance Th1 cell development compared to WT BMDCs. Furthermore, this response could be reversed by LFA-1 blockade. Our data point to two related but distinct mechanisms by which PTPN22 regulates LFA-1 dependent signals to enhance Th1 development, highlighting how perturbations to PTPN22 function over time to regulate the balance of the immune response.
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http://dx.doi.org/10.1016/j.jaut.2018.07.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6198113PMC
November 2018

Efficient Enrichment of Gene-Modified Primary T Cells via CCR5-Targeted Integration of Mutant Dihydrofolate Reductase.

Mol Ther Methods Clin Dev 2018 Jun 5;9:347-357. Epub 2018 Apr 5.

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

Targeted gene therapy strategies utilizing homology-driven repair (HDR) allow for greater control over transgene integration site, copy number, and expression-significant advantages over traditional vector-mediated gene therapy with random genome integration. However, the relatively low efficiency of HDR-based strategies limits their clinical application. Here, we used HDR to knock in a mutant dihydrofolate reductase (mDHFR) selection gene at the gene-edited CCR5 locus in primary human CD4 T cells and selected for mDHFR-modified cells in the presence of methotrexate (MTX). Cells were transfected with CCR5-megaTAL nuclease mRNA and transduced with adeno-associated virus containing an mDHFR donor template flanked by CCR5 homology arms, leading to up to 40% targeted gene insertion. Clinically relevant concentrations of MTX led to a greater than 5-fold enrichment for mDHFR-modified cells, which maintained a diverse TCR repertoire over the course of expansion and drug selection. Our results demonstrate that mDHFR/MTX-based selection can be used to enrich for gene-modified T cells , paving the way for analogous approaches to increase the percentage of HIV-resistant, autologous CD4 T cells infused into HIV patients, and/or for selection of gene-edited T cells for the treatment of cancer.
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http://dx.doi.org/10.1016/j.omtm.2018.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054698PMC
June 2018

αv Integrins regulate germinal center B cell responses through noncanonical autophagy.

J Clin Invest 2018 08 20;128(9):4163-4178. Epub 2018 Aug 20.

Immunology Program, Benaroya Research Institute at Virginia Mason, Seattle, Washington, USA.

Germinal centers (GCs) are major sites of clonal B cell expansion and generation of long-lived, high-affinity antibody responses to pathogens. Signaling through TLRs on B cells promotes many aspects of GC B cell responses, including affinity maturation, class switching, and differentiation into long-lived memory and plasma cells. A major challenge for effective vaccination is identifying strategies to specifically promote GC B cell responses. Here, we have identified a mechanism of regulation of GC B cell TLR signaling, mediated by αv integrins and noncanonical autophagy. Using B cell-specific αv-KO mice, we show that loss of αv-mediated TLR regulation increased GC B cell expansion, somatic hypermutation, class switching, and generation of long-lived plasma cells after immunization with virus-like particles (VLPs) or antigens associated with TLR ligand adjuvants. Furthermore, targeting αv-mediated regulation increased the magnitude and breadth of antibody responses to influenza virus vaccination. These data therefore identify a mechanism of regulation of GC B cells that can be targeted to enhance antibody responses to vaccination.
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http://dx.doi.org/10.1172/JCI99597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6118577PMC
August 2018

TACI deletion protects against progressive murine lupus nephritis induced by BAFF overexpression.

Kidney Int 2018 10 12;94(4):728-740. Epub 2018 Jun 12.

Seattle Children's Research Institute, Seattle, Washington, USA; Department of Pediatrics, University of Washington School of Medicine, Seattle, Washington, USA. Electronic address:

B cells are known to promote the pathogenesis of systemic lupus erythematosus (SLE) via the production of pathogenic anti-nuclear antibodies. However, the signals required for autoreactive B cell activation and the immune mechanisms whereby B cells impact lupus nephritis pathology remain poorly understood. The B cell survival cytokine B cell activating factor of the TNF Family (BAFF) has been implicated in the pathogenesis of SLE and lupus nephritis in both animal models and human clinical studies. Although the BAFF receptor has been predicted to be the primary BAFF family receptor responsible for BAFF-driven humoral autoimmunity, in the current study we identify a critical role for signals downstream of Transmembrane Activator and CAML Interactor (TACI) in BAFF-dependent lupus nephritis. Whereas transgenic mice overexpressing BAFF develop progressive membranoproliferative glomerulonephritis, albuminuria and renal dysfunction, TACI deletion in BAFF-transgenic mice provided long-term (about 1 year) protection from renal disease. Surprisingly, disease protection in this context was not explained by complete loss of glomerular immune complex deposits. Rather, TACI deletion specifically reduced endocapillary, but not mesangial, immune deposits. Notably, although excess BAFF promoted widespread breaks in B cell tolerance, BAFF-transgenic antibodies were enriched for RNA- relative to DNA-associated autoantigen reactivity. These RNA-associated autoantibody specificities were specifically reduced by TACI or Toll-like receptor 7 deletion. Thus, our study provides important insights into the autoantibody specificities driving proliferative lupus nephritis, and suggests that TACI inhibition may be novel and effective treatment strategy in lupus nephritis.
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http://dx.doi.org/10.1016/j.kint.2018.03.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6151274PMC
October 2018

Rapid immune reconstitution of SCID-X1 canines after G-CSF/AMD3100 mobilization and in vivo gene therapy.

Blood Adv 2018 05;2(9):987-999

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

Hematopoietic stem-cell gene therapy is a promising treatment of X-linked severe combined immunodeficiency disease (SCID-X1), but currently, it requires recipient conditioning, extensive cell manipulation, and sophisticated facilities. With these limitations in mind, we explored a simpler therapeutic approach to SCID-X1 treatment by direct IV administration of foamy virus (FV) vectors in the canine model. FV vectors were used because they have a favorable integration site profile and are resistant to serum inactivation. Here, we show improved efficacy of our in vivo gene therapy platform by mobilization with granulocyte colony-stimulating factor (G-CSF) and AMD3100 before injection of an optimized FV vector incorporating the human phosphoglycerate kinase enhancerless promoter. G-CSF/AMD3100 mobilization before FV vector delivery accelerated kinetics of CD3 lymphocyte recovery, promoted thymopoiesis, and increased immune clonal diversity. Gene-corrected T lymphocytes exhibited a normal CD4:CD8 ratio and a broad T-cell receptor repertoire and showed restored γC-dependent signaling function. Treated animals showed normal primary and secondary antibody responses to bacteriophage immunization and evidence for immunoglobulin class switching. These results demonstrate safety and efficacy of an accessible, portable, and translatable platform with no conditioning regimen for the treatment of SCID-X1 and other genetic diseases.
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http://dx.doi.org/10.1182/bloodadvances.2018016451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5942001PMC
May 2018

Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells.

Mol Ther 2018 02 22;26(2):456-467. Epub 2017 Nov 22.

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

The ability to engineer primary human B cells to differentiate into long-lived plasma cells and secrete a de novo protein may allow the creation of novel plasma cell therapies for protein deficiency diseases and other clinical applications. We initially developed methods for efficient genome editing of primary B cells isolated from peripheral blood. By delivering CRISPR/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complexes under conditions of rapid B cell expansion, we achieved site-specific gene disruption at multiple loci in primary human B cells (with editing rates of up to 94%). We used this method to alter ex vivo plasma cell differentiation by disrupting developmental regulatory genes. Next, we co-delivered RNPs with either a single-stranded DNA oligonucleotide or adeno-associated viruses containing homologous repair templates. Using either delivery method, we achieved targeted sequence integration at high efficiency (up to 40%) via homology-directed repair. This method enabled us to engineer plasma cells to secrete factor IX (FIX) or B cell activating factor (BAFF) at high levels. Finally, we show that introduction of BAFF into plasma cells promotes their engraftment into immunodeficient mice. Our results highlight the utility of genome editing in studying human B cell biology and demonstrate a novel strategy for modifying human plasma cells to secrete therapeutic proteins.
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http://dx.doi.org/10.1016/j.ymthe.2017.11.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5835153PMC
February 2018

Protein tyrosine phosphatase PTPN22 is dispensable for dendritic cell antigen processing and promotion of T-cell activation by dendritic cells.

PLoS One 2017 17;12(10):e0186625. Epub 2017 Oct 17.

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

The PTPN22R620W single nucleotide polymorphism increases the risk of developing multiple autoimmune diseases including type 1 diabetes, rheumatoid arthritis and lupus. PTPN22 is highly expressed in antigen presenting cells (APCs) where the expression of the murine disease associated variant orthologue (Ptpn22R619W) is reported to dysregulate pattern recognition receptor signalling in dendritic cells (DCs) and promote T-cell proliferation. Because T-cell activation is dependent on DC antigen uptake, degradation and presentation, we analysed the efficiency of these functions in splenic and GM-CSF bone marrow derived DC from wild type (WT), Ptpn22-/- or Ptpn22R619W mutant mice. Results indicated no differential ability of DCs to uptake antigen via macropinocytosis or receptor-mediated endocytosis. Antigen degradation and presentation was also equal as was WT T-cell conjugate formation and subsequent T-cell proliferation. Despite the likely presence of multiple phosphatase-regulated pathways in the antigen uptake, processing and presentation pathways that we investigated, we observed that Ptpn22 and the R619W autoimmune associated variant were dispensable. These important findings indicate that under non-inflammatory conditions there is no requirement for Ptpn22 in DC dependent antigen uptake and T-cell activation. Our findings reveal that perturbations in antigen uptake and processing, a fundamental pathway determining adaptive immune responses, are unlikely to provide a mechanism for the risk associated with the Ptpn22 autoimmune associated polymorphism.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186625PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5645108PMC
October 2017

Protein tyrosine phosphatase PTPN22 regulates IL-1β dependent Th17 responses by modulating dectin-1 signaling in mice.

Eur J Immunol 2018 02 20;48(2):306-315. Epub 2017 Oct 20.

Academic Department of Rheumatology, Centre for Inflammation Biology and Cancer Immunology, Faculty of Life Sciences and Medicine, King's College London, London, UK.

A single nucleotide polymorphism within the PTPN22 gene is a strong genetic risk factor predisposing to the development of multiple autoimmune diseases. PTPN22 regulates Syk and Src family kinases downstream of immuno-receptors. Fungal β-glucan receptor dectin-1 signals via Syk, and dectin-1 stimulation induces arthritis in mouse models. We investigated whether PTPN22 regulates dectin-1 dependent immune responses. Bone marrow derived dendritic cells (BMDCs) generated from C57BL/6 wild type (WT) and Ptpn22 mutant mice, were pulsed with OVA and the dectin-1 agonist curdlan and co-cultured in vitro with OT-II T-cells or adoptively transferred into OT-II mice, and T-cell responses were determined by immunoassay. Dectin-1 activated Ptpn22 BMDCs enhanced T-cell secretion of IL-17 in vitro and in vivo in an IL-1β dependent manner. Immunoblotting revealed that compared to WT, dectin-1 activated Ptpn22 BMDCs displayed enhanced Syk and Erk phosphorylation. Dectin-1 activation of BMDCs expressing Ptpn22 (the mouse orthologue of human PTPN22 ) also resulted in increased IL-1β secretion and T-cell dependent IL-17 responses, indicating that in the context of dectin-1 Ptpn22 operates as a loss-of-function variant. These findings highlight PTPN22 as a novel regulator of dectin-1 signals, providing a link between genetically conferred perturbations of innate receptor signaling and the risk of autoimmune disease.
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http://dx.doi.org/10.1002/eji.201747092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5859948PMC
February 2018