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1.
Biol Methods Protoc ; 8(1): bpad018, 2023.
Article in English | MEDLINE | ID: mdl-37720517

ABSTRACT

Viral vectors are commonly used to introduce genetic material into cells to modify cell function for a variety of purposes. Manufacture of those modified viruses may use a variety of cell types to generate high titers of viral particles; one of the most common being HEK293 cells. These cells have been modified into different lines aimed at satisfying specific use cases. HEK293T cells, for example, have been modified to include the SV40 large T antigen. Efficient viral particle production by HEK293T cells requires the maintenance of favorable cell culture conditions during expansion and transfection. This protocol describes the use of the Quantum® hollow-fiber bioreactor (HFB) system for the automated expansion of HEK293T cells, and the results derived using the protocol described herein were not compared with those from tissue culture flasks or other expansion platforms, as the parameters described are unique to Quantum's hollow fiber cell expansion environment. The purpose of this protocol is to help users of Quantum to focus on relevant parameters of expansion in the HFB milieu and to provide guidelines for a successful expansion of HEK293T cells in the Quantum system. The steps provided have been optimized to reliably control environmental factors related to glucose, lactate, and pH. Data reflecting this consistency are provided along with procedural time points reflected in text and figure formats.

2.
Mucosal Immunol ; 16(3): 357-372, 2023 06.
Article in English | MEDLINE | ID: mdl-37088263

ABSTRACT

Differences in transcriptomes, transcription factor usage, and function have identified T follicular helper 2 (Tfh2) cells and T helper 2 (Th2) cells as distinct clusters of differentiation 4+",(CD4) T-cell subsets in settings of type-2 inflammation. Although the transcriptional programs driving Th2 cell differentiation and cytokine production are well defined, dependence on these classical Th2 programs by Tfh2 cells is less clear. Using cytokine reporter mice in combination with transcription factor inference analysis, the b-Zip transcription factor c-Maf and its targets were identified as an important regulon in both Th2 and Tfh2 cells. Conditional deletion of c-Maf in T cells confirmed its importance in type-2 cytokine expression by Th2 and Tfh2 cells. However, while c-Maf was not required for Th2-driven helminth clearance or lung eosinophilia, it was required for Tfh2-driven Immunoglobulin E production and germinal center formation. This differential regulation of cell-mediated and humoral immunity by c-Maf was a result of redundant pathways in Th2 cells that were absent in Tfh2 cells, and c-Maf-specific mechanisms in Tfh2 cells that were absent in Th2 cells. Thus, despite shared expression by Tfh2 and Th2 cells, c-Maf serves as a unique regulator of Tfh2-driven humoral hallmarks during type-2 immunity.


Subject(s)
Helminthiasis , Th2 Cells , Mice , Animals , Gene Expression Regulation , Transcription Factors/metabolism , Cytokines/metabolism , Gene Expression , Th1 Cells
3.
Cytotherapy ; 24(11): 1148-1157, 2022 11.
Article in English | MEDLINE | ID: mdl-36031522

ABSTRACT

BACKGROUND AIMS: Modular automation is a flexible and reliable option to build the foundation of a new or evolving process or to introduce automation to a process that is already established. Herein the authors demonstrate that modular automation provides both high-quality and high-yield T-cell products. METHODS: Cells from three individual donors collected on an automated continuous flow centrifugation system were successfully expanded in a functionally closed, automated, perfusion-based hollow fiber bioreactor. These cells were then prepared for cryopreservation in an automated closed-system device that maintains temperature and aliquots a mixed cell product and cryoprotectant into product bags. Cell product bags were thawed and expanded in flasks. Samples taken throughout this manufacturing process were analyzed for cell phenotype, exhaustion markers and functionality. The proportion of CD4+ and CD8+ T cells was maintained through each step, from pre-expansion and post-expansion to immediately after thaw and 24 h after thaw. RESULTS: Interestingly, phenotypic markers such as CD45RO, CD45RA and CCR7 evolved throughout the process and stem-like memory T cells emerged as the predominant phenotype in the clinically relevant 24-h post-thaw sample. CONCLUSIONS: Modular automation supported the generation of stem-like memory T cells that were not terminally exhausted and were able to produce effector cytokines upon restimulation.


Subject(s)
Cryopreservation , Memory T Cells , Cell- and Tissue-Based Therapy , Cytokines , Leukocyte Common Antigens , Perfusion , Receptors, CCR7
4.
PLoS Pathog ; 17(6): e1009602, 2021 06.
Article in English | MEDLINE | ID: mdl-34106992

ABSTRACT

The CD4+ T cell response is critical to host protection against helminth infection. How this response varies across different hosts and tissues remains an important gap in our understanding. Using IL-4-reporter mice to identify responding CD4+ T cells to Nippostrongylus brasiliensis infection, T cell receptor sequencing paired with novel clustering algorithms revealed a broadly reactive and clonally diverse CD4+ T cell response. While the most prevalent clones and clonotypes exhibited some tissue selectivity, most were observed to reside in both the lung and lung-draining lymph nodes. Antigen-reactivity of the broader repertoires was predicted to be shared across both tissues and individual mice. Transcriptome, trajectory, and chromatin accessibility analysis of lung and lymph-node repertoires revealed three unique but related populations of responding IL-4+ CD4+ T cells consistent with T follicular helper, T helper 2, and a transitional population sharing similarity with both populations. The shared antigen reactivity of lymph node and lung repertoires combined with the adoption of tissue-specific gene programs allows for the pairing of cellular and humoral responses critical to the orchestration of anti-helminth immunity.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Strongylida Infections/immunology , Animals , Lung/immunology , Lymph Nodes/immunology , Mice , Nippostrongylus , Receptors, Antigen, T-Cell, alpha-beta/immunology , Single-Cell Analysis
5.
J Immunol ; 205(8): 2039-2045, 2020 10 15.
Article in English | MEDLINE | ID: mdl-32917785

ABSTRACT

Early thymic progenitors (ETPs) are bone marrow-derived hematopoietic stem cells that remain multipotent and give rise to a variety of lineage-specific cells. Recently, we discovered a subset of murine ETPs that expresses the IL-4Rα/IL-13Rα1 heteroreceptor (HR) and commits only to the myeloid lineage. This is because IL-4/IL-13 signaling through the HR inhibits their T cell potential and enacts commitment of HR+ETPs to thymic resident CD11c+CD8α+ dendritic cells (DCs). In this study, we discovered that HR+-ETP-derived DCs function as APCs in the thymus and promote deletion of myelin-reactive T cells. Furthermore, this negative T cell selection function of HR+-ETP-derived DCs sustains protection against experimental allergic encephalomyelitis, a mouse model for human multiple sclerosis. These findings, while shedding light on the intricacies underlying ETP lineage commitment, reveal a novel, to our knowledge, function by which IL-4 and IL-13 cytokines condition thymic microenvironment to rheostat T cell selection and fine-tune central tolerance.


Subject(s)
Dendritic Cells/immunology , Interleukin-13/immunology , Interleukin-4/immunology , Multiple Sclerosis/immunology , T-Lymphocytes/immunology , Thymus Gland/immunology , Animals , Cellular Microenvironment/genetics , Cellular Microenvironment/immunology , Dendritic Cells/pathology , Disease Models, Animal , Interleukin-13/genetics , Interleukin-4/genetics , Mice , Mice, Knockout , Multiple Sclerosis/genetics , Multiple Sclerosis/pathology , T-Lymphocytes/pathology , Thymus Gland/pathology
6.
Front Immunol ; 11: 1594, 2020.
Article in English | MEDLINE | ID: mdl-32793230

ABSTRACT

Soil-transmitted helminths represent a major global health burden with infections and infection-related comorbidities causing significant reductions in the quality of life for individuals living in endemic areas. Repeated infections and chronic colonization by these large extracellular worms in mammals led to the evolution of type-2 immunity characterized by the production of the type-2 cytokines interleukin (IL)-4, IL-5, and IL-13. Although a number of adaptive and innate immune cells produce type-2 cytokines, a key cellular source in the context of helminth infection is group 2 innate lymphoid cells (ILC2s). ILC2s promote mucosal barrier homeostasis, integrity, and repair by rapidly responding to epithelial cues in mucosal tissues. Though tissue-resident ILC2s (nILC2s) have been studied in detail over the last decade, considerably less is known with regard to a subset of inflammatory ILC2s (iILC2s) that migrate to the lungs of mice early after Nippostrongylus brasiliensis infection and are potent early producers of type-2 cytokines. This review will discuss the relationship and differences between nILC2s and iILC2s that establish their unique roles in anti-helminth immunity. We have placed particular emphasis on studies investigating iILC2 origin, function, and their potential long-term contribution to tissue-resident ILC2 reservoirs in settings of helminth infection.


Subject(s)
Helminthiasis/immunology , Helminths/immunology , Lymphocyte Subsets/immunology , Lymphocytes/immunology , Animals , Cytokines/immunology , Global Health , Helminthiasis/epidemiology , Inflammation , Lymphocyte Subsets/cytology , Lymphocytes/cytology , Nippostrongylus/immunology
7.
Sci Immunol ; 5(43)2020 01 10.
Article in English | MEDLINE | ID: mdl-31924686

ABSTRACT

A transitory, interleukin-25 (IL-25)-responsive, group 2 innate lymphoid cell (ILC2) subset induced during type 2 inflammation was recently identified as iILC2s. This study focuses on understanding the significance of this population in relation to tissue-resident nILC2s in the lung and intestine. RNA-sequencing and pathway analysis revealed the AP-1 superfamily transcription factor BATF (basic leucine zipper transcription factor, activating transcription factor-like) as a potential modulator of ILC2 cell fate. Infection of BATF-deficient mice with Nippostrongylus brasiliensis showed a selective defect in IL-25-mediated helminth clearance and a corresponding loss of iILC2s in the lung characterized as IL-17RBhigh, KLRG1high, BATFhigh, and Arg1low BATF deficiency selectively impaired iILC2s because it had no impact on tissue-resident nILC2 frequency or function. Pulmonary-associated iILC2s migrated to the lung after infection, where they represented an early source of IL-4 and IL-13. Although the composition of ILC2s in the small intestine was distinct from those in the lung, their frequency and IL-13 expression remained dependent on BATF, which was also required for optimal goblet and tuft cell hyperplasia. Findings support IL-25-responsive ILC2s as early sentinels of mucosal barrier integrity.


Subject(s)
Basic-Leucine Zipper Transcription Factors/immunology , Cytokines/immunology , Lymphocytes/immunology , Nippostrongylus , Strongylida Infections/immunology , Allergens/immunology , Animals , Basic-Leucine Zipper Transcription Factors/genetics , Female , Intestine, Small/immunology , Lung/immunology , Male , Mice, Transgenic , Pyroglyphidae/immunology
8.
Crit Rev Immunol ; 40(5): 407-411, 2020.
Article in English | MEDLINE | ID: mdl-33463951

ABSTRACT

This brief review is written in memory of Eli Sercarz, a colleague who among many achievements, pioneered and revitalized early-life immunity, a field that is of high relevance to child health. For a long time, the neonatal stage was viewed as a window during which exposure to antigen (Ag) induces immune tolerance. In early 1990, however, it was discovered that the newborn mouse given Ag on the day of birth develops immunity when challenged later with the same Ag. But, these secondary responses displayed a deficit in T-helper (Th)1 cells and excess Th2 lymphocytes. Such discoveries explain the perceived tolerance of Ags given at the neonatal stage and correlate the paucity of effective neonatal vaccines and vulnerability to allergic reactions. Analyzing the mechanisms underlying neonatal Th1 cell deficits revealed a complex developmental interaction between Ag-presenting cells and the cytokines that they produced. This culminated into limited interleukin (IL)-12 in the environment and up-regulation of IL-13Rα1 expression and its association with IL-4Rα on the surface of Th1 cells. After Ag re-exposure, Th2 cells produce IL-4 and -13. Both bind to the heteroreceptor on Th1 cell surfaces and trigger their death. Usually, cytokines promote growth of T cells, but in this case IL-4 and -13 stimulate production of interferon regulatory factor 1 (IRF-1). IRF-1 translocates from nucleus to cytoplasm and stimulates apoptotic machinery that terminates Th1 cells. This suggests that vaccine formulations that could elevate IL-12 production are likely to counter IL-13Rα1 expression, preserve Th1 cells, and potentiate vaccine efficacy in neonates.


Subject(s)
Th1 Cells , Vaccines , Animals , Cytokines , Humans , Immune Tolerance , Mice , Th2 Cells
9.
J Immunol ; 202(11): 3173-3186, 2019 06 01.
Article in English | MEDLINE | ID: mdl-30996000

ABSTRACT

Early life immune responses are deficient in Th1 lymphocytes that compromise neonatal vaccination. We found that IL-4 and IL-13 engage a developmentally expressed IL-4Rα/IL-13Rα1 heteroreceptor to endow IFN regulatory factor 1 (IRF-1) with apoptotic functions, which redirect murine neonatal Th1 reactivation to cell death. IL-4/IL-13-induced STAT6 phosphorylation serves to enhance IRF-1 transcription and promotes its egress from the nucleus. In the cytoplasm, IRF-1 can no longer serve as an anti-viral transcription factor but, instead, colocalizes with Bim and instigates the mitochondrial, or intrinsic, death pathway. The new pivotal function of IRF-1 in the death of neonatal Th1 cells stems from the ability of its gene to bind STAT6 for enhanced transcription and the proficiency of its protein to precipitate Bim-driven apoptosis. This cytokine-induced, IRF-1-mediated developmental death network weakens neonatal Th1 responses during early life vaccination and increases susceptibility to viral infection.


Subject(s)
Interferon Regulatory Factor-1/metabolism , Interleukin-13/metabolism , Interleukin-4/metabolism , Th1 Cells/immunology , Viral Vaccines/immunology , Virus Diseases/immunology , Animals , Animals, Newborn , Apoptosis , Bcl-2-Like Protein 11/metabolism , Disease Resistance , Humans , Immunity , Infant, Newborn , Interferon Regulatory Factor-1/genetics , Interleukin-13 Receptor alpha1 Subunit/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , STAT6 Transcription Factor/metabolism , Signal Transduction
10.
J Immunol ; 201(10): 2947-2958, 2018 11 15.
Article in English | MEDLINE | ID: mdl-30291166

ABSTRACT

Recently we reported that IL-4 and IL-13 signaling in murine early thymic progenitors (ETPs) expressing the heteroreceptor (HR) comprising IL-4 receptor α (IL-4Rα) and IL-13 receptor α 1 (IL-13Rα1) activate STAT6 and inhibit ETP maturation potential toward T cells. In this study, we asked whether IL-4 and IL-13 signaling through the HR mobilizes other STAT molecules to shape ETP fate decision. The findings indicate that HR+ ETPs undergoing cytokine signaling display increased STAT1, but not STAT3, phosphorylation in addition to STAT6 activation. In parallel, the ETPs had a STAT1-dependent heightened expression of IRF-8, a transcription factor essential for development of CD8α+ dendritic cells (DCs). Interestingly, STAT1 phosphorylation and IRF-8 upregulation, which are independent of STAT6 activation, guided ETP maturation toward myeloid cells with a CD8α+ DC phenotype. Furthermore, these CD8α+ DCs display a thymic resident phenotype, as they did not express SIRPα, a molecule presumed to be involved in cell migration. These findings suggest that IL-4 and IL-13 cytokine-induced HR signaling provides a double-edged sword that simultaneously blocks T cell lineage potential but advances myeloid maturation that could impact T cell selection and central tolerance.


Subject(s)
Cell Differentiation/immunology , Dendritic Cells/cytology , Interleukin-13/metabolism , Interleukin-4/metabolism , Thymocytes/cytology , Animals , Central Tolerance/immunology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Female , Interferon Regulatory Factors/immunology , Interferon Regulatory Factors/metabolism , Interleukin-13/immunology , Interleukin-4/immunology , Mice , Mice, Knockout , STAT1 Transcription Factor/immunology , STAT1 Transcription Factor/metabolism , STAT6 Transcription Factor/immunology , STAT6 Transcription Factor/metabolism , Thymocytes/immunology , Thymocytes/metabolism
11.
Cell Immunol ; 331: 130-136, 2018 09.
Article in English | MEDLINE | ID: mdl-29929727

ABSTRACT

IL-13 receptor alpha 1 (IL-13Rα1) associates with IL-4Rα to form a functional IL-4Rα/IL-13Rα1 heteroreceptor (HR) through which both IL-4 and IL-13 signal. Recently, HR expression was associated with the development of M2 type macrophages which function as antigen presenting cells (APCs). Herein, we show that a subset of thymic resident dendritic cells (DCs) expressing high CD11b (CD11bhi) and intermediate CD11c (CD11cint) arise in HR-sufficient but not HR-deficient mice. These DCs, which originate from the bone marrow are able to take up Ag from the peritoneum, traffic through the spleen and the lymph nodes and carry it to the thymus. In addition, since the DCs are able to present Ag to T cells, express high levels of the costimulatory molecule CD24, and comprise a CD8α+ subset, it is likely that the cells contribute to T cell development and perhaps negative selection of self-reactive lymphocytes.


Subject(s)
Antigen Presentation/immunology , Cell Movement/immunology , Dendritic Cells/immunology , Interleukin-13 Receptor alpha1 Subunit/immunology , Thymus Gland/immunology , Animals , CD11c Antigen/immunology , CD11c Antigen/metabolism , CD24 Antigen/immunology , CD24 Antigen/metabolism , Dendritic Cells/metabolism , Interleukin-13 Receptor alpha1 Subunit/genetics , Interleukin-13 Receptor alpha1 Subunit/metabolism , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
12.
J Immunol ; 199(8): 2767-2776, 2017 10 15.
Article in English | MEDLINE | ID: mdl-28893952

ABSTRACT

Early thymic progenitors (ETPs) are endowed with diverse potencies and can give rise to myeloid and lymphoid lineage progenitors. How the thymic environment guides ETP commitment and maturation toward a specific lineage remains obscure. We have previously shown that ETPs expressing the heteroreceptor (HR) comprising IL-4Rα and IL-13Rα1 give rise to myeloid cells but not T cells. In this article, we show that signaling through the HR inhibits ETP maturation to the T cell lineage but enacts commitment toward the myeloid cells. Indeed, HR+ ETPs, but not HR- ETPs, exhibit activated STAT6 transcription factor, which parallels with downregulation of Notch1, a critical factor for T cell development. Meanwhile, the myeloid-specific transcription factor C/EBPα, usually under the control of Notch1, is upregulated. Furthermore, in vivo inhibition of STAT6 phosphorylation restores Notch1 expression in HR+ ETPs, which regain T lineage potential. In addition, upon stimulation with IL-4 or IL-13, HR- ETPs expressing virally transduced HR also exhibit STAT6 phosphorylation and downregulation of Notch1, leading to inhibition of lymphoid, but not myeloid, lineage potential. These observations indicate that environmental cytokines play a role in conditioning ETP lineage choice, which would impact T cell development.


Subject(s)
Interleukin-13 Receptor alpha1 Subunit/metabolism , Interleukin-13/metabolism , Interleukin-4/metabolism , Precursor Cells, T-Lymphoid/physiology , Receptors, Cell Surface/metabolism , T-Lymphocytes/physiology , Thymus Gland/immunology , Animals , CCAAT-Enhancer-Binding Proteins/metabolism , Cell Differentiation , Cell Lineage , Cells, Cultured , Interleukin-13 Receptor alpha1 Subunit/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Cells/physiology , Receptors, Cell Surface/genetics , STAT6 Transcription Factor/metabolism , Signal Transduction
13.
J Immunol ; 199(7): 2236-2248, 2017 10 01.
Article in English | MEDLINE | ID: mdl-28801358

ABSTRACT

IL-4 and IL-13 have been defined as anti-inflammatory cytokines that can counter myelin-reactive T cells and modulate experimental allergic encephalomyelitis. However, it is not known whether endogenous IL-4 and IL-13 contribute to the maintenance of peripheral tolerance and whether their function is coordinated with T regulatory cells (Tregs). In this study, we used mice in which the common cytokine receptor for IL-4 and IL-13, namely the IL-4Rα/IL-13Rα1 (13R) heteroreceptor (HR), is compromised and determined whether the lack of signaling by endogenous IL-4 and IL-13 through the HR influences the function of effector Th1 and Th17 cells in a Treg-dependent fashion. The findings indicate that mice-deficient for the HR (13R-/-) are more susceptible to experimental allergic encephalomyelitis than mice sufficient for the HR (13R+/+) and develop early onset and more severe disease. Moreover, Th17 cells from 13R-/- mice had reduced ability to convert to Th1 cells and displayed reduced sensitivity to suppression by Tregs relative to Th17 effectors from 13R+/+ mice. These observations suggest that IL-4 and IL-13 likely operate through the HR and influence Th17 cells to convert to Th1 cells and to acquire increased sensitivity to suppression, leading to control of immune-mediated CNS inflammation. These previously unrecognized findings shed light on the intricacies underlying the contribution of cytokines to peripheral tolerance and control of autoimmunity.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/immunology , Interleukin-13 Receptor alpha1 Subunit/immunology , Receptors, Cell Surface/immunology , T-Lymphocytes, Regulatory/immunology , Th17 Cells/immunology , Animals , Cytokines/biosynthesis , Cytokines/immunology , Disease Models, Animal , Immune Tolerance , Interleukin-13/biosynthesis , Interleukin-13/metabolism , Interleukin-13 Receptor alpha1 Subunit/deficiency , Interleukin-13 Receptor alpha1 Subunit/genetics , Interleukin-4/biosynthesis , Interleukin-4/metabolism , Mice , Mice, Inbred C57BL , Receptors, Cell Surface/deficiency , Receptors, Cell Surface/genetics , Signal Transduction , Th1 Cells/immunology
14.
J Immunol ; 199(3): 894-902, 2017 08 01.
Article in English | MEDLINE | ID: mdl-28646042

ABSTRACT

Type 1 diabetes (T1D) manifests when the insulin-producing pancreatic ß cells are destroyed as a consequence of an inflammatory process initiated by lymphocytes of the immune system. The NOD mouse develops T1D spontaneously and serves as an animal model for human T1D. The IL-4Rα/IL-13Rα1 heteroreceptor (HR) serves both IL-4 and IL-13 cytokines, which are believed to function as anti-inflammatory cytokines in T1D. However, whether the HR provides a responsive element to environmental (i.e., physiologic) IL-4/IL-13 in the regulation of peripheral tolerance and the development of T1D has yet to be defined. In this study, NOD mice deficient for the HR have been generated by means of IL-13Rα1 gene disruption and used to determine whether such deficiency affects the development of T1D. Surprisingly, the findings indicate that NOD mice lacking the HR (13R-/-) display resistance to T1D as the rise in blood glucose level and islet inflammation were significantly delayed in these HR-deficient relative to HR-sufficient (13R+/+) mice. In fact, the frequency and spleen-to-pancreas dynamics of both Th1 and Th17 cells were affected in 13R-/- mice. This is likely due to an increase in the frequency of mTGFß+Foxp3int regulatory T cells and the persistence of CD206+ macrophages in the pancreas as both types of cells confer resistance to T1D upon transfer to 13R+/+ mice. These findings reveal new insights as to the role environmental IL-4/IL-13 and the HR play in peripheral tolerance and the development of T1D.


Subject(s)
Diabetes Mellitus, Type 1/immunology , Interleukin-13 Receptor alpha1 Subunit/metabolism , Receptors, Cell Surface/metabolism , Adoptive Transfer , Animals , Blood Glucose , Disease Models, Animal , Insulin-Secreting Cells/immunology , Interleukin-13/immunology , Interleukin-13 Receptor alpha1 Subunit/deficiency , Interleukin-13 Receptor alpha1 Subunit/genetics , Interleukin-13 Receptor alpha1 Subunit/immunology , Interleukin-4/immunology , Lectins, C-Type/immunology , Macrophages/immunology , Mannose Receptor , Mannose-Binding Lectins/immunology , Mice , Mice, Inbred NOD , Pancreas/cytology , Pancreas/immunology , Receptors, Cell Surface/genetics , Receptors, Cell Surface/immunology , T-Lymphocytes, Regulatory/immunology , Th1 Cells/immunology , Th17 Cells/immunology
15.
J Immunol ; 197(9): 3554-3565, 2016 11 01.
Article in English | MEDLINE | ID: mdl-27671108

ABSTRACT

To contain autoimmunity, pathogenic T cells must be eliminated or diverted from reaching the target organ. Recently, we defined a novel form of T cell tolerance whereby treatment with Ag downregulates expression of the chemokine receptor CXCR3 and prevents diabetogenic Th1 cells from reaching the pancreas, leading to suppression of type 1 diabetes (T1D). This report defines the signaling events underlying Ag-induced chemokine receptor-mediated tolerance. Specifically, we show that the mammalian target of rapamycin complex 1 (mTORC1) is a major target for induction of CXCR3 downregulation and crippling of Th1 cells. Indeed, Ag administration induces upregulation of programmed death-ligand 1 on dendritic cells in a T cell-dependent manner. In return, programmed death-ligand 1 interacts with the constitutively expressed programmed death-1 on the target T cells and stimulates docking of Src homology 2 domain-containing tyrosine phosphatase 2 phosphatase to the cytoplasmic tail of programmed death-1. Active Src homology 2 domain-containing tyrosine phosphatase 2 impairs the signaling function of the PI3K/protein kinase B (AKT) pathway, leading to functional defect of mTORC1, downregulation of CXCR3 expression, and suppression of T1D. Thus, mTORC1 component of the metabolic pathway serves as a target for chemokine receptor-mediated T cell tolerance and suppression of T1D.


Subject(s)
Multiprotein Complexes/immunology , Receptors, CXCR3/metabolism , T-Lymphocytes/immunology , TOR Serine-Threonine Kinases/immunology , Animals , Antigens/immunology , Autoimmunity , B7-H1 Antigen/metabolism , Cells, Cultured , Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 1/therapy , Immune Tolerance , Immunomodulation , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Inbred NOD , Programmed Cell Death 1 Receptor/metabolism , Signal Transduction
16.
Biochem Biophys Res Commun ; 467(4): 766-70, 2015 Nov 27.
Article in English | MEDLINE | ID: mdl-26478433

ABSTRACT

Health policy precludes neonatal vaccination against influenza. Hence, morbidity and mortality are high under 6 months of age. Lactoferrin may activate diminished numbers of dysfunctional dendritic cells and reverse neonatal vaccine failures. Aluminum hydroxide/ALUM recruits neutrophils that secrete lactoferrin at deposition sites of antigen. We theorized lactoferrin + influenza antigen initiates an equivalent antibody response compared to ALUM. Three-day-old mice received subcutaneously 30 µg of H1N1 hemagglutinin + 200 µg of bovine lactoferrin versus hemagglutinin + ALUM. Controls received hemagglutinin, lactoferrin, or ALUM. After 21 days, sera measured anti-H1N1 (ELISA) and neutralizing antibody (plaque assays). ELISA detected equal antibody production with lactoferrin + hemagglutinin compared to hemagglutinin + ALUM; both sera also neutralized H1N1 virus at a 1:20 dilution (p < 0.01). Controls had no anti-H1N1 antibody. Neonates given lactoferrin had no anaphylaxis when challenged four weeks later. Lactoferrin is a safe and effective adjuvant for inducing antibody against influenza in neonates.


Subject(s)
Adjuvants, Immunologic/pharmacology , Lactoferrin/pharmacology , Orthomyxoviridae Infections/immunology , Vaccination/methods , Aluminum Hydroxide/immunology , Aluminum Hydroxide/pharmacology , Animals , Animals, Newborn , Cattle , Dogs , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Hemagglutinin Glycoproteins, Influenza Virus/pharmacology , Influenza A Virus, H1N1 Subtype/pathogenicity , Lactoferrin/immunology , Madin Darby Canine Kidney Cells/virology , Mice, Inbred BALB C , Orthomyxoviridae Infections/prevention & control , Orthomyxoviridae Infections/virology
17.
Neonatology ; 108(2): 137-42, 2015.
Article in English | MEDLINE | ID: mdl-26159186

ABSTRACT

BACKGROUND: Eosinophils infiltrate intestinal tissue during necrotizing enterocolitis (NEC) and adult bowel diseases. We theorized that epithelial damage causes eosinophilic activation and recruitment at NEC onset. OBJECTIVE: We studied the relationship between persistent blood eosinophilia and medical or surgical complications during NEC. METHODS: NEC cases and controls at MU Children's Hospital (2008-2013) underwent review. A Likert scale measured NEC severity. We utilized an SPSS database for statistical analyses. RESULTS: Of 50 NEC cases, infants in group 1 (n = 15) had eosinophilia <2 days after onset and those in group 2 (n = 25) had NEC but no persistent eosinophilia. Group 3 (n = 46) consisted of controls, i.e. infants without NEC matched for birth weight and gestational age and group 4 (n = 4) of preterm infants with infection and ≤5 days of eosinophilia. Hematologic assessment defined persistent eosinophilia as ≥5% eosinophils for ≥5 days after NEC onset. Absolute eosinophil counts were 2 times higher in group 1 than in group 2 (p = 0.002). The mean duration of eosinophilia was 8 days in group 1 versus 1 day in group 2 (p < 0.001). A Likert score of NEC severity was 3-fold higher in group 1 than in group 2 (p < 0.001). Compared to group 2, group 1 infants were 8 times more likely to have hepatic fibrosis or intestinal strictures. CONCLUSIONS: Early persistent blood eosinophilia is not currently a predictor of complications after the onset of NEC. This biomarker identifies immature infants at a high risk for adverse outcomes during NEC convalescence.


Subject(s)
Cytokines/blood , Enterocolitis, Necrotizing/complications , Eosinophilia/blood , Infant, Premature/blood , Biomarkers/blood , Birth Weight , Databases, Factual , Female , Gestational Age , Humans , Infant, Newborn , Intensive Care Units, Neonatal , Male , Missouri , Prognosis , Retrospective Studies , Severity of Illness Index
18.
J Immunol ; 195(2): 507-18, 2015 Jul 15.
Article in English | MEDLINE | ID: mdl-26034171

ABSTRACT

Neonatal immunity exhibits weak Th1 but excessive Th2 responses, and the underlying mechanisms remain elusive. In this article, we show that neonatal basophils readily produce IL-4, a cytokine that proved to be pivotal in shaping the programs of both lymphocyte subsets. Besides promoting Th2 programs, IL-4 is captured by the IL-4 heteroreceptor (IL-4Rα/IL-13Rα1) expressed on dendritic cells and instigates IL-12 downregulation. Under these circumstances, differentiating Th1 cells upregulate IL-13Rα1, leading to an unusual expression of the heteroreceptor, which will serve as a death marker for these Th1 cells during rechallenge with Ag. The resulting Th1/Th2 imbalance impacts childhood immunity culminating in sensitivity to allergic reactions, susceptibility to microbial infection and perhaps poor efficacy of pediatric vaccines.


Subject(s)
Basophils/immunology , Dendritic Cells/immunology , Gene Expression Regulation, Developmental/immunology , Th1 Cells/immunology , Th2 Cells/immunology , Adoptive Transfer , Amino Acid Sequence , Animals , Animals, Newborn , Basophils/cytology , Basophils/drug effects , Basophils/transplantation , Dendritic Cells/cytology , Dendritic Cells/drug effects , Dendritic Cells/transplantation , Female , Immunity, Innate , Interleukin-12/genetics , Interleukin-12/immunology , Interleukin-13 Receptor alpha1 Subunit/genetics , Interleukin-13 Receptor alpha1 Subunit/immunology , Interleukin-4/genetics , Interleukin-4/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Molecular Sequence Data , Ovalbumin/pharmacology , Receptors, Cell Surface/genetics , Receptors, Cell Surface/immunology , Signal Transduction , Th1 Cells/cytology , Th1 Cells/drug effects , Th1 Cells/transplantation , Th2 Cells/cytology , Th2 Cells/drug effects , Th2 Cells/transplantation
19.
Curr Opin Pediatr ; 26(2): 146-50, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24503532

ABSTRACT

PURPOSE OF REVIEW: There is an intense interest among neonatal caregivers as to whether lactoferrin given enterally may reduce the incidence of necrotizing enterocolitis in preterm infants. This review presents scientific and clinical evidence that lactoferrin alleviates or prevents this life-threatening disease. RECENT FINDINGS: Preclinical studies in neonatal rats showed that lactoferrin given orally before enteral infection with pathogenic Escherichia coli reduced bacteremia and mortality. A multicentered clinical trial found that very low-birth weight preterm infants given bovine lactoferrin had a significant reduction in late-onset sepsis; there was also a trend towards a diminished incidence of necrotizing enterocolitis. Although multicentered trials of lactoferrin use in preterm infants are near completion, regulatory burdens required to bring lactoferrin to the bedside may limit its availability. SUMMARY: Extremely preterm infants should receive colostrum, a natural lactoferrin concentrate, immediately after birth and, ideally, continue on breast milk throughout the hospital stay. This practice appears well tolerated, but additional experience will tell us whether this practice reduces the prevalence of necrotizing enterocolitis.


Subject(s)
Colostrum , Enterocolitis, Necrotizing/prevention & control , Gastrointestinal Tract/immunology , Inflammation/immunology , Lactoferrin/therapeutic use , Sepsis/prevention & control , Animals , Enteral Nutrition , Enterocolitis, Necrotizing/immunology , Humans , Immunity, Innate , Incidence , Infant, Newborn , Infant, Premature , Infant, Premature, Diseases/prevention & control , Infant, Very Low Birth Weight , Lactoferrin/administration & dosage , Lactoferrin/immunology , Randomized Controlled Trials as Topic , Sepsis/immunology
20.
Eur J Immunol ; 44(3): 842-55, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24281978

ABSTRACT

In this study, we examined the role IL-13 receptor alpha 1 (IL-13Rα1) plays in macrophage differentiation and function. The findings indicate that IL-13Rα1 is expressed on the M2 but not on the M1 subset of macrophages and specifically heterodimerizes with the IL-4Rα chain to form a type II receptor, which controls the differentiation and function of these cells. Indeed, BM cells from IL-13Rα1(+/+) and IL-13Rα1(-/-) mice yield equivalent numbers of macrophages when cultured under M2 polarizing conditions. However, IL-13Rα1(-/-) BM cells yield a much higher number of macrophages than IL-13Rα1(+/+) BM cells when the differentiation is carried out under M1-polarizing conditions. Further analyses indicated that macrophages that express IL-13Rα1 also display surface markers associated with an M2 phenotype. In addition, the IL-13Rα1(+) macrophages were highly efficient in phagocytizing zymosan bioparticles both in vitro and in vivo, and supported differentiation of naïve T cells to a Th2 phenotype. Finally, when stimulated by IL-13, a cytokine that uses the heteroreceptor, the cells were able to phosphorylate STAT6 efficiently. These previously unrecognized findings indicate that IL-13Rα1 serves as a marker for M2 macrophages and the resulting heteroreceptor influences both their differentiation and function.


Subject(s)
Interleukin-13 Receptor alpha1 Subunit/metabolism , Macrophages/immunology , Macrophages/metabolism , Animals , Cell Differentiation/genetics , Cell Differentiation/immunology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Gene Expression , Immunophenotyping , Interleukin-13/pharmacology , Interleukin-13 Receptor alpha1 Subunit/genetics , Macrophages/cytology , Macrophages/drug effects , Mice , Mice, Knockout , Monocytes/immunology , Monocytes/metabolism , Phenotype , Phosphorylation/drug effects , Quantitative Trait, Heritable , STAT6 Transcription Factor/metabolism
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