ABSTRACT
Antiviral CD8+ T cell immunity depends on the integration of various contextual cues, but how antigen-presenting cells (APCs) consolidate these signals for decoding by T cells remains unclear. Here, we describe gradual interferon-α/interferon-ß (IFNα/ß)-induced transcriptional adaptations that endow APCs with the capacity to rapidly activate the transcriptional regulators p65, IRF1 and FOS after CD4+ T cell-mediated CD40 stimulation. While these responses operate through broadly used signaling components, they induce a unique set of co-stimulatory molecules and soluble mediators that cannot be elicited by IFNα/ß or CD40 alone. These responses are critical for the acquisition of antiviral CD8+ T cell effector function, and their activity in APCs from individuals infected with severe acute respiratory syndrome coronavirus 2 correlates with milder disease. These observations uncover a sequential integration process whereby APCs rely on CD4+ T cells to select the innate circuits that guide antiviral CD8+ T cell responses.
Subject(s)
Antiviral Agents , COVID-19 , Humans , Calibration , Antigen-Presenting Cells , CD8-Positive T-Lymphocytes , CD40 Antigens , Interferon-alpha , CD4-Positive T-LymphocytesABSTRACT
Poor maternal diet during pregnancy is a risk factor for severe lower respiratory infections (sLRIs) in the offspring, but the underlying mechanisms remain elusive. Here, we demonstrate that in mice a maternal low-fiber diet (LFD) led to enhanced LRI severity in infants because of delayed plasmacytoid dendritic cell (pDC) recruitment and perturbation of regulatory T cell expansion in the lungs. LFD altered the composition of the maternal milk microbiome and assembling infant gut microbiome. These microbial changes reduced the secretion of the DC growth factor Flt3L by neonatal intestinal epithelial cells and impaired downstream pDC hematopoiesis. Therapy with a propionate-producing bacteria isolated from the milk of high-fiber diet-fed mothers, or supplementation with propionate, conferred protection against sLRI by restoring gut Flt3L expression and pDC hematopoiesis. Our findings identify a microbiome-dependent Flt3L axis in the gut that promotes pDC hematopoiesis in early life and confers disease resistance against sLRIs.
Subject(s)
Microbiota , Respiratory Tract Infections , Animals , Female , Mice , Pregnancy , Dendritic Cells , Diet , PropionatesABSTRACT
Central to adaptive immunity is the interaction between the αß T cell receptor (TCR) and peptide presented by the major histocompatibility complex (MHC) molecule. Presumably reflecting TCR-MHC bias and T cell signaling constraints, the TCR universally adopts a canonical polarity atop the MHC. We report the structures of two TCRs, derived from human induced T regulatory (iT(reg)) cells, complexed to an MHC class II molecule presenting a proinsulin-derived peptide. The ternary complexes revealed a 180° polarity reversal compared to all other TCR-peptide-MHC complex structures. Namely, the iT(reg) TCR α-chain and ß-chain are overlaid with the α-chain and ß-chain of MHC class II, respectively. Nevertheless, this TCR interaction elicited a peptide-reactive, MHC-restricted T cell signal. Thus TCRs are not 'hardwired' to interact with MHC molecules in a stereotypic manner to elicit a T cell signal, a finding that fundamentally challenges our understanding of TCR recognition.
Subject(s)
Autoantigens/metabolism , Major Histocompatibility Complex/immunology , Receptors, Antigen, T-Cell/metabolism , Adaptive Immunity , Antigen Presentation , Autoantigens/chemistry , Autoantigens/genetics , Cells, Cultured , HLA-DR4 Antigen/chemistry , HLA-DR4 Antigen/genetics , HLA-DR4 Antigen/metabolism , Histocompatibility Antigens Class II/chemistry , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/metabolism , Humans , Major Histocompatibility Complex/genetics , Models, Molecular , Mutagenesis, Site-Directed , Proinsulin/chemistry , Proinsulin/genetics , Proinsulin/immunology , Protein Interaction Domains and Motifs , Receptors, Antigen, T-Cell/chemistry , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes, Regulatory/immunologyABSTRACT
Growing interest surrounds adoptive cellular therapies utilizing Natural Killer (NK) cells, which can be obtained from various sources, including umbilical cord blood (UCB) and adult peripheral blood (APB). Understanding NK cell receptor expression and diversity in such cellular sources will guide future therapeutic designs. We used a 20-color flow cytometry panel to compare unstimulated and cytokine-activated UCB and APB NK cells. Our analysis showed that UCB NK cells express slightly higher levels of the immune checkpoints PD-1, TIGIT, and CD96 compared to their APB counterparts. Unsupervised hierarchical clustering and dimensionality reduction analyses revealed enrichment in CD56neg as well as mature NKp46neg and CD56+ CD16+ NK cell populations in UCB whereas CD57+ terminally differentiated NK cells with variable expression of KIRs and CD16 were found in APB. These populations were conserved following stimulation with IL-12, IL-15, and IL-18. Cytokine stimulation was associated with the downregulation of TIGIT and CD16 on multiple NK cell subsets in UCB and APB. Among UCB CD16- NK cell populations, TIGIT+ NK cells produced more IFN-γ than their TIGIT- counterparts. Our data demonstrate higher immune checkpoint expression on UCB NK cells compared to APB. However, the expression of TIGIT immune checkpoint is not indicative of NK cell exhaustion.
Subject(s)
Fetal Blood , Killer Cells, Natural , Adult , Humans , Cytokines , Interleukin-12 , Flow Cytometry , CD56 AntigenABSTRACT
Next-generation humanised mouse models and single-cell RNA sequencing (scRNAseq) approaches enable in-depth studies into human immune cell biology. Here we used NSG-SGM3 mice engrafted with human umbilical cord haematopoietic stem cells to investigate how human immune cells respond to and/or are changed by traumatic spinal cord injury (SCI). We hypothesised that the use of such mice could help advance our understanding of spinal cord injury-induced immune depression syndrome (SCI-IDS), and also how human leukocytes change as they migrate from the circulation into the lesion site. Our scRNAseq experiments, supplemented by flow cytometry, demonstrate the existence of up to 11 human immune cell (sub-) types and/or states across the blood and injured spinal cord (7 days post-SCI) of humanised NSG-SGM3 mice. Further comparisons of human immune cell transcriptomes between naïve, sham-operated and SCI mice identified a total of 579 differentially expressed genes, 190 of which were 'SCI-specific' (that is, genes regulated only in response to SCI but not sham surgery). Gene ontology analysis showed a prominent downregulation of immune cell function under SCI conditions, including for T cell receptor signalling and antigen presentation, confirming the presence of SCI-IDS and the transcriptional signature of human leukocytes in association with this phenomenon. We also highlight the activating influence of the local spinal cord lesion microenvironment by comparing the transcriptomes of circulating versus infiltrated human immune cells; those isolated from the lesion site were enriched for genes relating to both immune cell activity and function (e.g., oxidative phosphorylation, T cell proliferation and antigen presentation). We lastly applied an integrated bioinformatics approach to determine where immune responses in humanised NSG-SGM3 mice appear congruent to the native responses of human SCI patients, and where they diverge. Collectively, our study provides a valuable resource and methodological framework for the use of these mice in translational research.
Subject(s)
Spinal Cord Diseases , Spinal Cord Injuries , Mice , Humans , Animals , Spinal Cord Injuries/metabolism , Leukocytes/pathology , Gene Expression , Sequence Analysis, RNAABSTRACT
Dendritic cells (DCs) are functionally diverse and are present in most adult tissues, but deep understanding of human DC biology is hampered by relatively small numbers of these in circulation and their short lifespan in human tissues. We built a transcriptional atlas of human DCs by combining samples from 14 expression profiling studies derived from 10 laboratories. We identified significant gene expression variation of DC subset-defining markers across tissue type and upon viral or bacterial stimulation. We further highlight critical gaps between in vitro-derived DC subsets and their in vivo counterparts and provide evidence that monocytes or cord blood progenitor in vitro-differentiated DCs fail to capture the repertoire of primary DC subsets or behaviors. In constructing a reference DC atlas, we provide an important resource for the community wishing to identify and annotate tissue-specific DC subsets from single-cell datasets, or benchmark new in vitro models of DC biology.
Subject(s)
Dendritic Cells , Monocytes , Humans , Dendritic Cells/metabolism , Cell Differentiation , BiologyABSTRACT
DEC-205 is a cell-surface receptor that transports bound ligands into the endocytic pathway for degradation or release within lysosomal endosomes. This receptor has been reported to bind a number of ligands, including keratin, and some classes of CpG oligodeoxynucleotides (ODN). In this study, we explore in detail the requirements for binding ODNs, revealing that DEC-205 efficiently binds single-stranded, phosphorothioated ODN of ≥14 bases, with preference for the DNA base thymidine, but with no requirement for a CpG motif. DEC-205 fails to bind double-stranded phosphodiester ODN, and thus does not bind the natural type of DNA found in mammals. The ODN binding preferences of DEC-205 result in strong binding of B class ODN, moderate binding to C class ODN, minimal binding to P class ODN, and no binding to A class ODN. Consistent with DEC-205 binding capacity, induction of serum IL-12p70 or activation of B cells by each class of ODN correlated with DEC-205 dependence in mice. Thus, the greater the DEC-205 binding capacity, the greater the dependence on DEC-205 for optimal responses. Finally, by covalently linking a B class ODN that efficiently binds DEC-205, to a P class ODN that shows poor binding, we improved DEC-205 binding and increased adjuvancy of the hybrid ODN. The hybrid ODN efficiently enhanced induction of effector CD8 T cells in a DEC-205-dependent manner. Furthermore, the hybrid ODN induced robust memory responses, and was particularly effective at promoting the development of liver tissue-resident memory T cells.
Subject(s)
Adjuvants, Immunologic , Oligodeoxyribonucleotides , Animals , Dendritic Cells , Interleukin-12 , Liver , MiceABSTRACT
The endothelial adhesion protein E-selectin/CD62E is not required for leukocyte homing, unlike closely related family member P-selectin/CD62P. As transmigration through the endothelium is one of the first steps in generating a local immune response, we hypothesized that E-selectin may play additional roles in the early stages of immune activation. We found contact with E-selectin, but not P-selectin or vascular cell adhesion molecule 1 (CD106), induced phosphorylation of protein kinase B (AKT) and nuclear factor-κB in mouse bone marrow-derived macrophages (BMDMs) in vitro. This occurred within 15 min of E-selectin contact and was dependent on phosphatidylinositol-3 kinase activity. Binding to E-selectin activated downstream proteins including mammalian target of rapamycin, p70 ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1. Functionally, adhesion to E-selectin induced upregulation of CD86 expression and CCL2 secretion. We next asked whether contact with E-selectin impacts further BMDM stimulation. We found enhanced secretion of both interleukin (IL)-10 and CCL2, but not tumor necrosis factor or IL-6 in response to lipopolysaccharide (LPS) stimulation after adhesion to E-selectin. Importantly, adhesion to E-selectin did not polarize BMDMs to one type of response but enhanced both arginase activity and nitric oxide production following IL-4 or LPS stimulation, respectively. In cultured human monocytes, adhesion to E-selectin similarly induced phosphorylation of AKT. Finally, when E-selectin was blocked in vivo in mice, thioglycollate-elicited macrophages showed reduced CD86 expression, validating our in vitro studies. Our results imply functions for E-selectin beyond homing and suggest that E-selectin plays an early role in priming and amplifying innate immune responses.
Subject(s)
E-Selectin , Proto-Oncogene Proteins c-akt , Animals , Cell Adhesion , Cells, Cultured , Endothelium, Vascular , Macrophages , Mice , TOR Serine-Threonine KinasesABSTRACT
Mice reconstituted with human hematopoietic stem cells are valuable models to study aspects of the human immune system in vivo. We describe a humanized mouse model (hu mice) in which fully functional human CD141+ and CD1c+ myeloid and CD123+ plasmacytoid dendritic cells (DC) develop from human cord blood CD34+ cells in immunodeficient mice. CD141+ DC are the human equivalents of murine CD8+ /CD103+ DC which are essential for the induction of tumor-inhibitory cytotoxic T lymphocyte responses, making them attractive targets to exploit for the development of new cancer immunotherapies. We used CD34+ -engrafted NSG-A2 mice to investigate activation of DC subsets by synthetic dsRNA or ssRNA analogs polyinosinic-polycytidylic acid/poly I:C and Resiquimod/R848, agonists for TLR3 and TLR8, respectively, both of which are expressed by CD141+ DC. Injection of hu mice with these agonists resulted in upregulation of costimulatory molecules CD80, CD83 and CD86 by CD141+ and CD1c+ DC alike, and their combination further enhanced expression of these molecules by both subsets. When combined, poly I:C and R848 enhanced serum levels of key cytokines associated with cross-presentation and the induction of cytotoxic T lymphocyte responses including IFN-α, IFN-ß, IL-12 and CXCL10. These data advocate a combination of poly I:C and R848 TLR agonists as means of activating human DC for immunotherapy.
Subject(s)
Antigens, CD1/metabolism , Antigens, Surface/metabolism , Dendritic Cells/immunology , Toll-Like Receptors/metabolism , Animals , Cell Differentiation/drug effects , Cytokines/blood , Dendritic Cells/drug effects , Humans , Imidazoles/pharmacology , Liver/drug effects , Liver/metabolism , Lung/drug effects , Lung/metabolism , Lymphoid Tissue/drug effects , Lymphoid Tissue/metabolism , Mice , Poly I-C/pharmacology , Toll-Like Receptors/agonists , Up-Regulation/drug effectsABSTRACT
Cross-presentation is the mechanism by which exogenous Ag is processed for recognition by CD8(+) T cells. Murine CD8α(+) DCs are specialized at cross-presenting soluble and cellular Ag, but in humans this process is poorly characterized. In this study, we examined uptake and cross-presentation of soluble and cellular Ag by human blood CD141(+) DCs, the human equivalent of mouse CD8α(+) DCs, and compared them with human monocyte-derived DCs (MoDCs) and blood CD1c(+) DC subsets. MoDCs were superior in their capacity to internalize and cross-present soluble protein whereas CD141(+) DCs were more efficient at ingesting and cross-presenting cellular Ag. Whilst cross-presentation by CD1c(+) DCs and CD141(+) DCs was dependent on the proteasome, and hence cytosolic translocation, cross-presentation by MoDCs was not. Inhibition of endosomal acidification enhanced cross-presentation by CD1c(+) DCs and MoDCs but not by CD141(+) DCs. These data demonstrate that CD1c(+) DCs, CD141(+) DCs, and MoDCs are capable of cross-presentation; however, they do so via different mechanisms. Moreover, they demonstrate that human CD141(+) DCs, like their murine CD8α(+) DC counterparts, are specialized at cross-presenting cellular Ag, most likely mediated by an enhanced capacity to ingest cellular Ag combined with subtle changes in lysosomal pH during Ag processing and use of the cytosolic pathway.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Endocytosis , Phosphoproteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Viral Matrix Proteins/metabolism , Antigens, CD1/metabolism , Antigens, Surface/metabolism , Blood Circulation , Cell Line , Cross-Priming , Humans , Monocytes/immunology , Necrosis , Protein Transport , Solubility , ThrombomodulinABSTRACT
We established a humanized mouse model incorporating FLT3-ligand (FLT3-L) administration after hematopoietic cell reconstitution to investigate expansion, phenotype, and function of human dendritic cells (DC). FLT3-L increased numbers of human CD141(+) DC, CD1c(+) DC, and, to a lesser extent, plasmacytoid DC (pDC) in the blood, spleen, and bone marrow of humanized mice. CD1c(+) DC and CD141(+) DC subsets were expanded to a similar degree in blood and spleen, with a bias toward expansion of the CD1c(+) DC subset in the bone marrow. Importantly, the human DC subsets generated after FLT3-L treatment of humanized mice are phenotypically and functionally similar to their human blood counterparts. CD141(+) DC in humanized mice express C-type lectin-like receptor 9A, XCR1, CADM1, and TLR3 but lack TLR4 and TLR9. They are major producers of IFN-λ in response to polyinosinic-polycytidylic acid but are similar to CD1c(+) DC in their capacity to produce IL-12p70. Although all DC subsets in humanized mice are efficient at presenting peptide to CD8(+) T cells, CD141(+) DC are superior in their capacity to cross-present protein Ag to CD8(+) T cells following activation with polyinosinic-polycytidylic acid. CD141(+) DC can be targeted in vivo following injection of Abs against human DEC-205 or C-type lectin-like receptor 9A. This model provides a feasible and practical approach to dissect the function of human CD141(+) and CD1c(+) DC and evaluate adjuvants and DC-targeting strategies in vivo.
Subject(s)
Adjuvants, Immunologic/pharmacology , Antigens, CD1/metabolism , Antigens, Surface/metabolism , Dendritic Cells/drug effects , Dendritic Cells/immunology , Glycoproteins/metabolism , Membrane Proteins/pharmacology , Adjuvants, Immunologic/administration & dosage , Animals , Antigen Presentation/immunology , Antigens, CD/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Adhesion Molecule-1 , Cell Adhesion Molecules/metabolism , Female , Humans , Immunoglobulins/metabolism , Interferon-gamma/metabolism , Interleukin-12/metabolism , Lectins, C-Type/immunology , Lectins, C-Type/metabolism , Lymphocyte Activation/immunology , Membrane Proteins/administration & dosage , Mice , Mice, Inbred NOD , Mice, SCID , Minor Histocompatibility Antigens , Poly I-C/immunology , Receptors, Cell Surface/immunology , Receptors, Chemokine/metabolism , Thrombomodulin , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 4/metabolism , Toll-Like Receptor 9/metabolismABSTRACT
The method of choice for the development of new vaccines is to target distinct dendritic cell subsets with antigen in vivo and to harness their function in situ to enhance cell-mediated immunity or induce tolerance to specific antigens. The innate functions of dendritic cells themselves may also be targeted by inhibitors or activators that would target a specific function such as interferon production, potentially important in autoimmune disease and chronic viral infections. Importantly targeting dendritic cells requires detailed knowledge of both the surface phenotype and function of each dendritic cell subset, including how they may respond to different types of vaccine adjuvants, their ability to produce soluble mediators and to process and present antigens and induce priming of naïve T cells. This review summarizes our knowledge of the functional attributes of the human dendritic cell subsets in the steady state and upon activation and their roles in human disease.
Subject(s)
Antigen Presentation/immunology , Dendritic Cells/immunology , Immune System Diseases/immunology , Immunity, Cellular/immunology , T-Lymphocytes/immunology , Antigens, CD1/immunology , Antigens, CD1/metabolism , Cytokines/immunology , Cytokines/metabolism , Dendritic Cells/metabolism , Glycoproteins/immunology , Glycoproteins/metabolism , Humans , Infections/immunology , Lymphocyte Activation/immunologyABSTRACT
Immunodeficient mice bearing human immune systems, or "humanized" chimeric mice, are widely used in basic research, along with the preclinical stages of drug development. Nonobese diabetic-severe combined immunodeficiency (NOD-SCID) IL2Rγnull (NSG) mice expressing human stem cell factor, granulocyte-macrophage colony stimulating factor, and interleukin-3 (NSG-SGM3) support robust development of human myeloid cells and T cells but have reduced longevity due to the development of fatal hemophagocytic lymphohistiocytosis (HLH). Here, we describe an optimized protocol for development of human immune chimerism in NSG-SGM3 mice. We demonstrate that efficient human CD45+ reconstitution can be achieved and HLH delayed by engraftment of neonatal NSG-SGM3 with low numbers of human umbilical cord-derived CD34+ hematopoietic stem cells in the absence of preconditioning irradiation.
Subject(s)
Hematopoietic Stem Cell Transplantation , Lymphohistiocytosis, Hemophagocytic , Mice , Humans , Animals , Infant, Newborn , Lymphohistiocytosis, Hemophagocytic/therapy , Mice, Inbred NOD , Mice, SCID , Hematopoietic Stem Cells , Antigens, CD34 , T-LymphocytesABSTRACT
Objectives: Immunotherapies targeting natural killer (NK) cell receptors have shown promise against leukaemia. Unfortunately, cancer immunosuppressive mechanisms that alter NK cell phenotype prevent such approaches from being successful. The study utilises advanced cytometry to examine how cancer immunosuppressive pathways affect NK cell phenotypic changes in clinical samples. Methods: In this study, we conducted a high-dimensional examination of the cell surface expression of 16 NK cell receptors in paediatric patients with acute myeloid leukaemia and acute lymphoblastic leukaemia, as well as in samples of non-age matched adult peripheral blood (APB) and umbilical cord blood (UCB). An unsupervised analysis was carried out in order to identify NK cell populations present in paediatric leukaemias. Results: We observed that leukaemia NK cells clustered together with UCB NK cells and expressed relatively higher levels of the NKG2A receptor compared to APB NK cells. In addition, CD56dimCD16+CD57- NK cells lacking NKG2A expression were mainly absent in paediatric leukaemia patients. However, CD56br NK cell populations expressing high levels of NKG2A were highly represented in paediatric leukaemia patients. NKG2A expression on leukaemia NK cells was found to be positively correlated with the expression of its ligand, suggesting that the NKG2A-HLA-E interaction may play a role in modifying NK cell responses to leukaemia cells. Conclusion: We provide an in-depth analysis of NK cell populations in paediatric leukaemia patients. These results support the development of immunotherapies targeting immunosuppressive receptors, such as NKG2A, to enhance innate immunity against paediatric leukaemia.
ABSTRACT
Human blood myeloid DCs can be subdivided into CD1c (BDCA-1)(+) and CD141 (BDCA-3)(+) subsets that display unique gene expression profiles, suggesting specialized functions. CD1c(+) DCs express TLR4 while CD141(+) DCs do not, thus predicting that these two subsets have differential capacities to respond to Escherichia coli. We isolated highly purified CD1c(+) and CD141(+) DCs and compared them to in vitro generated monocyte-derived DCs (MoDCs) following stimulation with whole E. coli. As expected, MoDCs produced high levels of the proinflammatory cytokines TNF, IL-6, and IL-12, were potent inducers of Th1 responses, and processed E. coli-derived Ag. In contrast, CD1c(+) DCs produced only low levels of TNF, IL-6, and IL-12 and instead produced high levels of the anti-inflammatory cytokine IL-10 and regulatory molecules IDO and soluble CD25. Moreover, E. coli-activated CD1c(+) DCs suppressed T-cell proliferation in an IL-10-dependent manner. Contrary to their mouse CD8(+) DC counterparts, human CD141(+) DCs did not phagocytose or process E. coli-derived Ag and failed to secrete cytokines in response to E. coli. These data demonstrate substantial differences in the nature of the response of human blood DC subsets to E. coli.
Subject(s)
Antigens, Surface/analysis , Dendritic Cells/immunology , Escherichia coli/immunology , Interleukin-10/biosynthesis , Myeloid Cells/immunology , Antigens, CD1 , Dendritic Cells/metabolism , Glycoproteins , Humans , Interleukin-10/metabolism , Lymphocyte Activation , Phenotype , T-Lymphocytes/immunology , ThrombomodulinABSTRACT
Hypoglycemia in type 1 diabetes associates with changes in the pancreatic islet α cells, where the receptor for advanced glycation end products (RAGE) is highly expressed. This study compared islet RAGE expression in donors without diabetes, those at risk of, and those with type 1 diabetes. Laser-dissected islets were subject to RNA bioinformatics and adjacent pancreatic tissue were assessed by confocal microscopy. We found that islets from type 1 diabetes donors had differential expression of the RAGE gene (AGER) and its correlated genes, based on glucagon expression. Random forest machine learning revealed that AGER was the most important predictor for islet glucagon levels. Conversely, a generalized linear model identified that glucagon expression could be predicted by expression of RAGE signaling molecules, its ligands and enzymes that create or clear RAGE ligands. Confocal imaging co-localized RAGE, its ligands and signaling molecules to the α cells. Half of the type 1 diabetes cohort comprised of adolescents and a patient with history of hypoglycemia-all showed an inverse relationship between glucagon and RAGE. These data confirm an association between glucagon and islet RAGE, its ligands and signaling pathways in type 1 diabetes, which warrants functional investigation into a role for RAGE in hypoglycemia.
Subject(s)
Diabetes Mellitus, Type 1 , Glucagon-Secreting Cells , Hypoglycemia , Receptor for Advanced Glycation End Products , Adolescent , Humans , Diabetes Mellitus, Type 1/genetics , Glucagon , Glucagon-Secreting Cells/metabolism , Glycation End Products, Advanced/metabolism , Ligands , Receptor for Advanced Glycation End Products/metabolismABSTRACT
Immunotherapy is a promising new treatment for patients with advanced prostate and ovarian cancer, but its application is limited by the lack of suitable target antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTL). Human kallikrein 4 (KLK4) is a member of the kallikrein family of serine proteases that is significantly overexpressed in malignant versus healthy prostate and ovarian tissue, making it an attractive target for immunotherapy. We identified a naturally processed, HLA-A*0201-restricted peptide epitope within the signal sequence region of KLK4 that induced CTL responses in vitro in most healthy donors and prostate cancer patients tested. These CTL lysed HLA-A*0201+ KLK4 + cell lines and KLK4 mRNA-transfected monocyte-derived dendritic cells. CTL specific for the HLA-A*0201-restricted KLK4 peptide were more readily expanded to a higher frequency in vitro compared to the known HLA-A*0201-restricted epitopes from prostate cancer antigens; prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA) and prostatic acid phosphatase (PAP). These data demonstrate that KLK4 is an immunogenic molecule capable of inducing CTL responses and identify it as an attractive target for prostate and ovarian cancer immunotherapy.
Subject(s)
Antigens, Neoplasm/metabolism , Kallikreins/metabolism , Peptide Fragments/metabolism , Prostatic Neoplasms/immunology , T-Lymphocytes, Cytotoxic/metabolism , Adult , Aged , Antigens, Neoplasm/immunology , Cell Proliferation , Computational Biology , Dendritic Cells/immunology , Female , HLA-A2 Antigen/metabolism , Humans , Immunodominant Epitopes/genetics , Kallikreins/immunology , Lymphocyte Activation , Male , Middle Aged , Peptide Fragments/immunology , Prostatic Neoplasms/pathology , Protein Sorting Signals/genetics , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/pathologyABSTRACT
The distribution and function of the C-type lectin Mincle has not previously been investigated in human cells, although mouse models have demonstrated a non-redundant role for Mincle in the host response to fungal infections. This study identified an unusual pattern of reciprocal expression of Mincle on peripheral blood monocytes or neutrophils isolated from the same donor. Expression on monocytes was inversely correlated with phagocytosis and yeast killing, but was necessary for the induction of inflammatory cytokines in response to ex vivo Candida challenge. In contrast, Mincle expression on neutrophils was associated with phagocytic and candidacidal potential of those cells. Candida challenge upregulated Mincle expression but only in Mincle+ cells. These data highlight species-specific differences between the regulation of Mincle expression in mouse and man. Reciprocal expression of Mincle modified the candidacidal potential of monocytes or neutrophils, suggesting it may also polarize the type of host response to fungal infection.
Subject(s)
Candida albicans/immunology , Lectins, C-Type/immunology , Monocytes/immunology , Neutrophils/immunology , Receptors, Immunologic/immunology , Candida albicans/physiology , Cell Polarity/immunology , Cells, Cultured , Flow Cytometry , Host-Pathogen Interactions/immunology , Humans , Interleukin-1beta/immunology , Interleukin-1beta/metabolism , Lectins, C-Type/metabolism , Lipopolysaccharides/immunology , Lipopolysaccharides/pharmacology , Microscopy, Fluorescence , Monocytes/drug effects , Monocytes/microbiology , Neutrophils/drug effects , Neutrophils/microbiology , Phagocytosis/immunology , Receptors, Immunologic/metabolism , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
INTRODUCTION: The use of dendritic cell (DC)-based cancer vaccines over three decades has shown them to be a safe therapeutic approach against a range of hematological and solid malignancies. However, underwhelming and inconsistent results from clinical trials have seen them move in and out of favor. The limitations of ex vivo generated monocyte-derived DC (MoDC) in these therapies provide a pointed explanation for the varying and somewhat disappointing clinical outcomes. The identification of a specialized role for the rare conventional type 1 dendritic cell (cDC1) subset in orchestrating tumor immunity via the initiation of CD8+ T cell responses has led to a new concept of targeting cDC1 as a therapeutic option to address the unmet need of enhancing the immune response in cancer patients. AREAS COVERED: This article reviews several current challenges and key opportunities associated with the development and use of next generation cDC1 cancer vaccines. EXPERT OPINION: Manipulation of cDC1 quantity and quality holds enormous potential to improve tumor immunogenicity, as already demonstrated in preclinical models. New technologies are rapidly advancing the understanding of cDC1 in human cancer patients and facilitating the generation of these extremely rare cells in vitro, providing feasible new approaches toward clinical translation.
Subject(s)
Cancer Vaccines , Neoplasms , Antigens, Neoplasm , CD8-Positive T-Lymphocytes , Cancer Vaccines/therapeutic use , Dendritic Cells , HumansABSTRACT
OBJECTIVES: DROSHA and DICER have central roles in the biogenesis of microRNAs (miRNAs). However, we previously showed that in the murine system, DROSHA has an alternate function where it directly recognises and cleaves protein-coding messenger (m)RNAs and this is critical for safeguarding the pluripotency of haematopoietic stem cells (HSCs). Maintenance of murine HSC function is dependent on DROSHA-mediated cleavage of two mRNAs, Myl9 and Todr1. The goal of this study is to determine whether this pathway is conserved in human HSCs. METHODS: DROSHA and DICER were knocked down in human cord blood CD34+ HSCs with short hairpin RNAs. The function of HSCs was analysed in vitro and in humanised mice. Analysis of mRNA cleavage was performed by capture of 5' phosphorylated RNAs. RESULTS: Consistent with murine HSCs, DROSHA knockdown impaired the differentiation of human HSCs in vitro and engraftment into humanised mice, whereas DICER knockdown had no impact. DROSHA cleaves the MYL9 mRNA in human HSCs and DROSHA deficiency resulted in the accumulation of the mRNA. However, ectopic expression of MYL9 did not impair human HSC function. We were unable to identify a human homolog of Todr1. CONCLUSION: A miRNA-independent function of DROSHA is critical for the function of human HSCs. DROSHA directly recognises and degrades mRNAs in humans HSCs. However, unlike in murine HSCs, the degradation of the MYL9 mRNA alone is not critical for human HSC function. Therefore, DROSHA must be inhibiting other targets and/or has another miRNA-independent function that is essential for safeguarding the pluripotency of human HSCs.