ABSTRACT
The quality of the adaptive immune response depends on the differentiation of distinct CD4(+) helper T cell subsets, and the magnitude of an immune response is controlled by CD4(+)Foxp3(+) regulatory T cells (Treg cells). However, how a tissue- and cell type-specific suppressor program of Treg cells is mechanistically orchestrated has remained largely unexplored. Through the use of Treg cell-specific gene targeting, we found that the suppression of allergic immune responses in the lungs mediated by T helper type 2 (TH2) cells was dependent on the activity of the protein kinase CK2. Genetic ablation of the ß-subunit of CK2 specifically in Treg cells resulted in the proliferation of a hitherto-unexplored ILT3(+) Treg cell subpopulation that was unable to control the maturation of IRF4(+)PD-L2(+) dendritic cells required for the development of TH2 responses in vivo.
Subject(s)
Casein Kinase II/immunology , T-Lymphocytes, Regulatory/immunology , Th2 Cells/immunology , Animals , CD4-Positive T-Lymphocytes/enzymology , CD4-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Cell Growth Processes/immunology , Cell Line , Dendritic Cells/enzymology , Dendritic Cells/immunology , Forkhead Transcription Factors/immunology , Humans , Hypersensitivity/blood , Hypersensitivity/immunology , Interferon Regulatory Factors/immunology , Leukocytes, Mononuclear/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Receptors, Cell Surface/immunology , T-Lymphocytes, Regulatory/enzymology , Th2 Cells/enzymologyABSTRACT
BNT162b2, a nucleoside-modified mRNA formulated in lipid nanoparticles that encodes the SARS-CoV-2 spike glycoprotein (S) stabilized in its prefusion conformation, has demonstrated 95% efficacy in preventing COVID-191. Here we extend a previous phase-I/II trial report2 by presenting data on the immune response induced by BNT162b2 prime-boost vaccination from an additional phase-I/II trial in healthy adults (18-55 years old). BNT162b2 elicited strong antibody responses: at one week after the boost, SARS-CoV-2 serum geometric mean 50% neutralizing titres were up to 3.3-fold above those observed in samples from individuals who had recovered from COVID-19. Sera elicited by BNT162b2 neutralized 22 pseudoviruses bearing the S of different SARS-CoV-2 variants. Most participants had a strong response of IFNγ+ or IL-2+ CD8+ and CD4+ T helper type 1 cells, which was detectable throughout the full observation period of nine weeks following the boost. Using peptide-MHC multimer technology, we identified several BNT162b2-induced epitopes that were presented by frequent MHC alleles and conserved in mutant strains. One week after the boost, epitope-specific CD8+ T cells of the early-differentiated effector-memory phenotype comprised 0.02-2.92% of total circulating CD8+ T cells and were detectable (0.01-0.28%) eight weeks later. In summary, BNT162b2 elicits an adaptive humoral and poly-specific cellular immune response against epitopes that are conserved in a broad range of variants, at well-tolerated doses.
Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , SARS-CoV-2/immunology , T-Lymphocytes/immunology , Adolescent , Adult , BNT162 Vaccine , CD8-Positive T-Lymphocytes/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/adverse effects , Epitopes, T-Lymphocyte/immunology , Female , Humans , Immunoglobulin G/immunology , Immunologic Memory , Interferon-gamma/immunology , Interleukin-2/immunology , Male , Middle Aged , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Th1 Cells/immunology , Young AdultABSTRACT
The additional author support information was erroneously omitted from the Supplementary Information. This has been corrected online.
ABSTRACT
Patients with glioblastoma currently do not sufficiently benefit from recent breakthroughs in cancer treatment that use checkpoint inhibitors1,2. For treatments using checkpoint inhibitors to be successful, a high mutational load and responses to neoepitopes are thought to be essential3. There is limited intratumoural infiltration of immune cells4 in glioblastoma and these tumours contain only 30-50 non-synonymous mutations5. Exploitation of the full repertoire of tumour antigens-that is, both unmutated antigens and neoepitopes-may offer more effective immunotherapies, especially for tumours with a low mutational load. Here, in the phase I trial GAPVAC-101 of the Glioma Actively Personalized Vaccine Consortium (GAPVAC), we integrated highly individualized vaccinations with both types of tumour antigens into standard care to optimally exploit the limited target space for patients with newly diagnosed glioblastoma. Fifteen patients with glioblastomas positive for human leukocyte antigen (HLA)-A*02:01 or HLA-A*24:02 were treated with a vaccine (APVAC1) derived from a premanufactured library of unmutated antigens followed by treatment with APVAC2, which preferentially targeted neoepitopes. Personalization was based on mutations and analyses of the transcriptomes and immunopeptidomes of the individual tumours. The GAPVAC approach was feasible and vaccines that had poly-ICLC (polyriboinosinic-polyribocytidylic acid-poly-L-lysine carboxymethylcellulose) and granulocyte-macrophage colony-stimulating factor as adjuvants displayed favourable safety and strong immunogenicity. Unmutated APVAC1 antigens elicited sustained responses of central memory CD8+ T cells. APVAC2 induced predominantly CD4+ T cell responses of T helper 1 type against predicted neoepitopes.
Subject(s)
Cancer Vaccines/immunology , Cancer Vaccines/therapeutic use , Glioblastoma/diagnosis , Glioblastoma/therapy , Precision Medicine/methods , Adult , Aged , Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/immunology , Epitopes, T-Lymphocyte/immunology , Female , Glioblastoma/immunology , HLA-A Antigens/immunology , Humans , Immunologic Memory/immunology , Male , Middle Aged , T-Lymphocytes, Helper-Inducer/immunology , Treatment OutcomeABSTRACT
T helper (Th)17 cells represent a unique subset of CD4+ T cells and are vital for clearance of extracellular pathogens including bacteria and fungi. However, Th17 cells are also involved in orchestrating autoimmunity. By employing quantitative surface proteomics, we found that the evolutionarily conserved prohibitins (PHB1/2) are highly expressed on the surface of both murine and human Th17 cells. Increased expression of PHBs at the cell surface contributed to enhanced CRAF/MAPK activation in Th17 cells. Targeting surface-expressed PHBs on Th17 cells with ligands such as Vi polysaccharide (Typhim vaccine) inhibited CRAF-MAPK pathway, reduced interleukin (IL)-17 expression and ameliorated disease pathology with an increase in FOXP3+-expressing Tregs in an animal model for multiple sclerosis (MS). Interestingly, we detected a CD4+ T cell population with high PHB1 surface expression in blood samples from MS patients in comparison with age- and sex-matched healthy subjects. Our observations suggest a pivotal role for the PHB-CRAF-MAPK signalling axis in regulating the polarization and pathogenicity of Th17 cells and unveil druggable targets in autoimmune disorders such as MS.
Subject(s)
Autoimmunity , Multiple Sclerosis/immunology , Repressor Proteins/immunology , Signal Transduction/immunology , Th17 Cells/immunology , Animals , Extracellular Signal-Regulated MAP Kinases/immunology , Forkhead Transcription Factors/immunology , HeLa Cells , Humans , Mice , Multiple Sclerosis/pathology , Prohibitins , Rickettsial Vaccines/pharmacology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathology , Th17 Cells/pathologyABSTRACT
Interferon-regulatory factor 4 (IRF4) is essential for the development of T helper 2 (Th2) and Th17 cells. Herein, we report that IRF4 is also crucial for the development and function of an interleukin-9 (IL-9)-producing CD4(+) T cell subset designated Th9. IRF4-deficient CD4(+) T cells failed to develop into IL-9-producing Th9 cells, and IRF4-specific siRNA inhibited IL-9 production in wild-type CD4(+) T cells. Chromatin-immunoprecipitation (ChIP) analyses revealed direct IRF4 binding to the Il9 promoter in Th9 cells. In a Th9-dependent asthma model, neutralization of IL-9 substantially ameliorated asthma symptoms. The relevance of these findings is emphasized by the fact that the induction of IL-9 production also occurs in human CD4(+) T cells accompanied by the upregulation of IRF4. Our data clearly demonstrate the central function of IRF4 in the development of Th9 cells and underline the contribution of this T helper cell subset to the pathogenesis of asthma.
Subject(s)
Interferon Regulatory Factors/immunology , Interleukin-9/immunology , T-Lymphocytes, Helper-Inducer/immunology , Animals , Asthma/genetics , Asthma/immunology , Cell Differentiation , Cells, Cultured , Humans , Interferon Regulatory Factors/deficiency , Interferon Regulatory Factors/genetics , Interferon Regulatory Factors/metabolism , Interleukin-9/biosynthesis , Interleukin-9/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Promoter Regions, Genetic , Protein Binding , RNA, Small Interfering/genetics , T-Lymphocytes, Helper-Inducer/cytologyABSTRACT
T helper 17 (TH17) cells represent a discrete TH cell subset instrumental in the immune response to extracellular bacteria and fungi. However, TH17 cells are considered to be detrimentally involved in autoimmune diseases like multiple sclerosis (MS). In contrast to TH17 cells, regulatory T (Treg) cells were shown to be pivotal in the maintenance of peripheral tolerance. Thus, the balance between Treg cells and TH17 cells determines the severity of a TH17 cell-driven disease and therefore is a promising target for treating autoimmune diseases. However, the molecular mechanisms controlling this balance are still unclear. Here, we report that pharmacological inhibition as well as genetic ablation of the protein kinase CK2 (CK2) ameliorates experimental autoimmune encephalomyelitis (EAE) severity and relapse incidence. Furthermore, CK2 inhibition or genetic ablation prevents TH17 cell development and promotes the generation of Treg cells. Molecularly, inhibition of CK2 leads to reduced STAT3 phosphorylation and strongly attenuated expression of the IL-23 receptor, IL-17, and GM-CSF. Thus, these results identify CK2 as a nodal point in TH17 cell development and suggest this kinase as a potential therapeutic target to treat TH17 cell-driven autoimmune responses.
Subject(s)
Casein Kinase II/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , T-Lymphocytes, Regulatory/immunology , Th17 Cells/immunology , Animals , Casein Kinase II/deficiency , Casein Kinase II/genetics , Encephalomyelitis, Autoimmune, Experimental/chemically induced , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/pathology , Forkhead Transcription Factors , Gene Expression Regulation , Granulocyte-Macrophage Colony-Stimulating Factor , Humans , Interleukin-17 , Mice , Mice, Inbred C57BL , Mice, Transgenic , Multiple Sclerosis/genetics , Multiple Sclerosis/immunology , Multiple Sclerosis/pathology , Myelin-Oligodendrocyte Glycoprotein , Peptide Fragments , Phosphorylation , Receptors, Interleukin , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/immunology , Severity of Illness Index , Signal Transduction , T-Lymphocytes, Regulatory/cytology , Th17 Cells/pathologyABSTRACT
Coevolution of ticks and the vertebrate immune system has led to the development of immunosuppressive molecules that prevent immediate response of skin-resident immune cells to quickly fend off the parasite. In this article, we demonstrate that the tick-derived immunosuppressor sialostatin L restrains IL-9 production by mast cells, whereas degranulation and IL-6 expression are both unaffected. In addition, the expression of IL-1ß and IRF4 is strongly reduced in the presence of sialostatin L. Correspondingly, IRF4- or IL-1R-deficient mast cells exhibit a strong impairment in IL-9 production, demonstrating the importance of IRF4 and IL-1 in the regulation of the Il9 locus in mast cells. Furthermore, IRF4 binds to the promoters of Il1b and Il9, suggesting that sialostatin L suppresses mast cell-derived IL-9 preferentially by inhibiting IRF4. In an experimental asthma model, mast cell-specific deficiency in IRF4 or administration of sialostatin L results in a strong reduction in asthma symptoms, demonstrating the immunosuppressive potency of tick-derived molecules.
Subject(s)
Cystatins/pharmacology , Immunity, Innate/drug effects , Immunosuppressive Agents/pharmacology , Interferon Regulatory Factors/immunology , Interleukin-9/immunology , Mast Cells/drug effects , Animals , Asthma/genetics , Asthma/immunology , Asthma/pathology , Binding Sites , Cell Degranulation/immunology , Cystatins/immunology , Gene Expression Regulation , Host-Parasite Interactions/immunology , Interferon Regulatory Factors/deficiency , Interferon Regulatory Factors/genetics , Interleukin-1beta/genetics , Interleukin-1beta/immunology , Interleukin-6/genetics , Interleukin-6/immunology , Interleukin-9/antagonists & inhibitors , Interleukin-9/genetics , Mast Cells/immunology , Mast Cells/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Promoter Regions, Genetic , Protein Binding , Receptors, Interleukin-1/genetics , Receptors, Interleukin-1/immunology , Signal Transduction , Transcription, GeneticABSTRACT
Asthma is a syndrome with different inflammatory phenotypes. Animal models have shown that, after sensitization and allergen challenge, Th2 and Th1 cells contribute to the development of allergic airway disease. We have previously demonstrated that naturally occurring regulatory T cells (nTregs) can only marginally suppress Th2-induced airway inflammation and airway hyperresponsiveness. In this study, we investigated nTreg-mediated suppression of Th2-induced and Th1-induced acute allergic airway disease. We demonstrate in vivo that nTregs exert their suppressive potency via cAMP transfer on Th2- and Th1-induced airway disease. A comparison of both phenotypes revealed that, despite similar cAMP transfers, Th1-driven airway hyperresponsiveness and inflammation are more susceptible to nTreg-dependent suppression, suggesting that potential nTreg-based therapeutic strategies might be more effective in patients with predominantly neutrophilic airway inflammation based on deregulated Th1 response.
Subject(s)
Bronchial Hyperreactivity/immunology , Disease Models, Animal , T-Lymphocytes, Regulatory/immunology , Th1 Cells/immunology , Th2 Cells/immunology , Acute Disease , Animals , Bronchial Hyperreactivity/pathology , Bronchial Hyperreactivity/prevention & control , Cells, Cultured , Coculture Techniques , Disease Susceptibility/immunology , Disease Susceptibility/pathology , Female , Immunity, Innate , Inflammation/immunology , Inflammation/pathology , Inflammation/prevention & control , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, Transgenic , T-Lymphocytes, Regulatory/pathology , Th1 Cells/pathology , Th2 Cells/pathologyABSTRACT
Next-generation sequencing (NGS) techniques provide unique prospects for in-depth transcriptome analyses. Nevertheless, the emerging and still growing knowledge about the large diversity and heterogeneity of cells that participate in immunological responses in a tissue- and micromilieu-specific manner calls for advanced isolation and sequencing methods for the accurate quantification of gene expression in small cell populations and even individual cells from any organ or tissue. One of the major limitations in performing transcriptome analyses of rare cell populations was and still is quality and quantity of RNA that often limits analyses of complex mixtures of immune cell populations. Here, we describe a protocol to isolate rare T cell populations from the lung and in particular the subsequent methods to isolate high-grade RNA in order to perform NGS-based transcriptome analyses.
Subject(s)
Gene Expression Profiling/methods , High-Throughput Nucleotide Sequencing/methods , RNA, Messenger/genetics , Single-Cell Analysis/methods , T-Lymphocytes/metabolism , Transcriptome , Animals , Cell Separation , DNA, Complementary/genetics , DNA, Complementary/metabolism , Gene Library , Genetic Heterogeneity , Lung/cytology , Lung/immunology , Lung/metabolism , Lymph Nodes/cytology , Lymph Nodes/immunology , Lymph Nodes/metabolism , Mice , RNA, Messenger/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/immunologyABSTRACT
OGG1 (8-oxoguanine-DNA glycosylase) is the major DNA repair glycosylase removing the premutagenic DNA base modification 8-oxo-7,8-dihydroguanine (8-oxoG) from the genome of mammalian cells. In addition, there is accumulating evidence that OGG1 and its substrate 8-oxoG might function in the regulation of certain genes, which could account for an attenuated immune response observed in Ogg1-/- mice in several settings. Indications for at least two different mechanisms have been obtained. Thus, OGG1 could either act as an ancillary transcription factor cooperating with the lysine-specific demethylase LSD1 or as an activator of small GTPases. Here, we analysed the activation by lipopolysaccaride (LPS) of primary splenocytes obtained from two different Ogg1-/- mouse strains. We found that the induction of TNF-α expression was reduced in splenocytes (in particular macrophages) of both Ogg1-/- strains. Notably, an inhibitor of LSD1, OG-L002, reduced the induction of TNF-α mRNA in splenocytes from wild-type mice to the level observed in splenocytes from Ogg1-/- mice and had no influence in the latter cells. In contrast, inhibitors of the MAP kinases p38 and JNK as well as the antioxidant N-acetylcysteine attenuated the LPS-stimulated TNF-α expression both in the absence and presence of OGG1. The free base 8-oxo-7,8-dihydroguanine had no influence on the TNF-α expression in the splenocytes. The data demonstrate that OGG1 plays a role in an LSD1-dependent pathway of LPS-induced macrophage activation in mice.
Subject(s)
DNA Glycosylases/immunology , Spleen/immunology , Tumor Necrosis Factor-alpha/genetics , Animals , DNA/metabolism , DNA Damage , DNA Glycosylases/metabolism , DNA Glycosylases/physiology , DNA Repair , Gene Expression Regulation , Guanine/analogs & derivatives , Guanine/metabolism , Macrophages/immunology , Macrophages/metabolism , Mice , Spleen/cytology , Spleen/metabolism , Transcription Factors/immunology , Transcription Factors/physiologyABSTRACT
Regulatory T cells (Treg) are essential for T cell homeostasis and maintenance of peripheral tolerance. They prevent activation of auto-reactive T effector cells (Teff) in the context of autoimmunity and allergy. Otherwise, Treg also inhibit effective immune responses against tumors. Besides a number of Treg-associated molecules such as Foxp3, CTLA-4 or GARP, known to play critical roles in Treg differentiation, activation and function, the involvement of additional regulatory elements is suggested. Herein, kinase activities seem to play an important role in Treg fine tuning. Nevertheless, our knowledge regarding the complex intracellular signaling pathways controlling phenotype and function of Treg is still limited and based on single kinase cascades so far. To gain a more comprehensive insight into the pathways determining Treg function we performed kinome profiling using a phosphorylation-based kinome array in human Treg at different activation stages compared to Teff. Here we have determined intriguing quantitative differences in both populations. Resting and activated Treg showed an altered pattern of CD28-dependent kinases as well as of those involved in cell cycle progression. Additionally, significant up-regulation of distinct kinases such as EGFR or CK2 in activated Treg but not in Teff not only resemble data we obtained in previous studies in the murine system but also suggest that those specific molecular activation patterns can be used for definition of the activation and functional state of human Treg. Taken together, detailed investigation of kinome profiles opens the possibility to identify novel molecular mechanisms for a better understanding of Treg biology but also for development of effective immunotherapies against unwanted T cell responses in allergy, autoimmunity and cancer.