| 1. |
- Cossarizza, A., et al.
(författare)
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Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)
- 2019
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Ingår i: European Journal of Immunology. - : Wiley. - 0014-2980 .- 1521-4141. ; 49:10, s. 1457-1973
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Tidskriftsartikel (refereegranskat)abstract
- These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
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| 3. |
- Aoun, M., et al.
(författare)
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Antigen-presenting autoreactive B cells activate regulatory T cells and suppress autoimmune arthritis in mice
- 2023
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Ingår i: Journal of Experimental Medicine. - Stockholm : Karolinska Institutet, Dept of Medical Biochemistry and Biophysics. - 0022-1007 .- 1540-9538. ; 220:11
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Tidskriftsartikel (refereegranskat)abstract
- B cells undergo several rounds of selection to eliminate potentially pathogenic autoreactive clones, but in contrast to T cells, evidence of positive selection of autoreactive B cells remains moot. Using unique tetramers, we traced natural autoreactive B cells (C1-B) specific for a defined triple-helical epitope on collagen type-II (COL2), constituting a sizeable fraction of the physiological B cell repertoire in mice, rats, and humans. Adoptive transfer of C1-B suppressed arthritis independently of IL10, separating them from IL10-secreting regulatory B cells. Single-cell sequencing revealed an antigen processing and presentation signature, including induced expression of CD72 and CCR7 as surface markers. C1-B presented COL2 to T cells and induced the expansion of regulatory T cells in a contact-dependent manner. CD72 blockade impeded this effect suggesting a new downstream suppressor mechanism that regulates antigen-specific T cell tolerization. Thus, our results indicate that autoreactive antigen-specific naive B cells tolerize infiltrating T cells against self-antigens to impede the development of tissue-specific autoimmune inflammation.
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| 4. |
- Johannesson, M, et al.
(författare)
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A resource for the simultaneous high-resolution mapping of multiple quantitative trait loci in rats: the NIH heterogeneous stock
- 2009
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Ingår i: Genome research. - : Cold Spring Harbor Laboratory. - 1088-9051. ; 19:1, s. 150-158
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Tidskriftsartikel (refereegranskat)abstract
- The laboratory rat (Rattus norvegicus) is a key tool for the study of medicine and pharmacology for human health. A large database of phenotypes for integrated fields such as cardiovascular, neuroscience, and exercise physiology exists in the literature. However, the molecular characterization of the genetic loci that give rise to variation in these traits has proven to be difficult. Here we show how one obstacle to progress, the fine-mapping of quantitative trait loci (QTL), can be overcome by using an outbred population of rats. By use of a genetically heterogeneous stock of rats, we map a locus contributing to variation in a fear-related measure (two-way active avoidance in the shuttle box) to a region on chromosome 5 containing nine genes. By establishing a protocol measuring multiple phenotypes including immunology, neuroinflammation, and hematology, as well as cardiovascular, metabolic, and behavioral traits, we establish the rat HS as a new resource for the fine-mapping of QTLs contributing to variation in complex traits of biomedical relevance.
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| 5. |
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| 6. |
- Li, QX, et al.
(författare)
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Rheumatoid arthritis sera antibodies to citrullinated collagen type II bind to joint cartilage
- 2022
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Ingår i: Arthritis research & therapy. - : Springer Science and Business Media LLC. - 1478-6362. ; 24:1, s. 257-
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Tidskriftsartikel (refereegranskat)abstract
- ObjectiveTo investigate the occurrence and frequency of anti-citrullinated protein antibodies (ACPA) to cyclic citrullinated type II collagen (COL2) epitope with a capacity to bind joint cartilage.MethodsLuminex immunoassay was used to analyze serum antibody reactivity to 10 COL2-citrullinated peptides (ACC10) and corresponding arginine peptide controls in rheumatoid arthritis (RA), osteoarthritis (OA), and healthy individuals’ cohorts. Top ten “promiscuous” sera (cross-reactive with all ACC10) and top ten “private” sera (restrictedly reactive with one ACC10 peptide) from RA and OA cohorts were selected. Enzyme-linked immunosorbent assay (ELISA) was used to detect response to native COL2. Sera were analyzed with naive and arthritic joints from DBA/1J mice by immunohistochemistry, using monoclonal ACPAs and COL2 reactive antibodies with human Fc as comparison. Staining specificity was confirmed with C1 (a major antibody epitope on COL2) mutated mice and competitive blocking with epitope-specific antibodies.ResultsAll patient sera bound ACC10 compared with control peptides but very few (3/40) bound native triple-helical COL2. Most sera (27/40) specifically bound to arthritic cartilage, whereas only one private RA serum bound to healthy cartilage. Despite very low titers, private sera from both RA and OA showed an epitope-specific response, documented by lack of binding to cartilage from C1-mutated mice and blocking binding to wild-type cartilage with a competitive monoclonal antibody. As a comparison, monoclonal ACPAs visualized typical promiscuous, or private reactivity to joint cartilage and other tissues.ConclusionACPA from RA and OA sera, reactive with citrullinated non-triple-helical COL2 peptides, can bind specifically to arthritic cartilage.
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| 7. |
- Michaëlsson, E., et al.
(författare)
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Macrophages, but not dendritic cells, present collagen to T cells
- 1995
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Ingår i: European Journal of Immunology. - : Wiley. - 0014-2980 .- 1521-4141. ; 25:8, s. 2234-2241
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Tidskriftsartikel (refereegranskat)abstract
- Dendritic cells, such as epidermal Langerhans cells, play a crucial role for the antigen-specific priming of T cells. We have addressed the question whether dendritic cells present collagen, a major protein component in tissues through which dendritic cells migrate, i.e. the basement membrane, dermis, and synovial tissue. Langerhans cells, spleen cells and peritoneal macrophages were compared for antigen-presenting capacity using a panel of mouse T cell hybridomas reactive with different determinants on type II collagen, myelin basic protein, ovalbumin and pepsin. Langerhans cells did not present any of the type II collagen determinants, unless the antigen was administered as a 15-mer peptide, but did present myelin basic protein, ovalbumin and pepsin. Spleen cells and peritoneal macrophages, in contrast, presented all type II collagen determinants. This biased antigen presentation was also observed when Langerhans cells were pulsed with antigen in vivo. The inability to present type II collagen is related to the collagen sequence as such, since both native type II collagen, type II collagen alpha chains, as well as a type II collagen determinant incorporated in type I collagen, were not presented by Langerhans cells. In addition, granulocyte/macrophage colony-stimulating factor-expanded blood dendritic cells displayed the same biased antigen presentation, suggesting that the inability to present collagen is not restricted to dendritic cells localized in epidermis. B cell-deficient mice could prime a type II collagen-reactive T cell response, thus excluding B cells as obligatory antigen-presenting cells for the priming of collagen-reactive T cells. We suggest that neither Langerhans cells nor B cells, but macrophages are the primary antigen-presenting cells in the immune response towards type II collagen.
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| 15. |
- Backlund, J., et al.
(författare)
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C57BL/6 mice need MHC class II Aq to develop collagen-induced arthritis dependent on autoreactive T cells
- 2013
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Ingår i: Annals of the Rheumatic Diseases. - : BMJ. - 0003-4967 .- 1468-2060. ; 72:7, s. 1225-1232
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Tidskriftsartikel (refereegranskat)abstract
- INTRODUCTION: Collagen-induced arthritis (CIA) has traditionally been performed in MHC class II A(q)-expressing mice, whereas most genetically modified mice are on the C57BL/6 background (expressing the b haplotype of the major histocompatibility complex (MHC) class II region). However, C57BL/6 mice develop arthritis after immunisation with chicken-derived collagen type II (CII), but arthritis susceptibility has been variable, and the immune specificity has not been clarified. OBJECTIVE: To establish a CIA model on the C57BL/6 background with a more predictable and defined immune response to CII. RESULTS: Both chicken and rat CII were arthritogenic in C57BL/6 mice provided they were introduced with high doses of Mycobacterium tuberculosis adjuvant. However, contaminating pepsin was strongly immunogenic and was essential for arthritis development. H-2(b)-restricted T cell epitopes on chicken or rat CII could not be identified, but expression of A(q) on the C57BL/6 background induced T cell response to the CII260-270 epitope, and also prolonged the arthritis to be more chronic. CONCLUSIONS: The putative (auto)antigen and its arthritogenic determinants in C57BL/6 mice remains undisclosed, questioning the value of the model for addressing T cell-driven pathological pathways in arthritis. To circumvent this impediment, we recommend MHC class II congenic C57BL/6N.Q mice, expressing A(q), with which T cell determinants have been thoroughly characterised.
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| 16. |
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| 17. |
- Bäcklund, A., et al.
(författare)
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Cystatin C influences the autoimmune but not inflammatory response to cartilage type II collagen leading to chronic arthritis development
- 2011
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Ingår i: Arthritis Research & Therapy. - : Springer Science and Business Media LLC. - 1478-6362. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- INTRODUCTION: Collagen-induced arthritis (CIA) is a mouse model for rheumatoid arthritis (RA) and is induced after immunization with type II collagen (CII). CIA, like RA, is an autoimmune disease leading to destruction of cartilage and joints, and both the priming and inflammatory phases have been suggested to be dependent on proteases. In particular, the cysteine proteases have been proposed to be detrimental to the arthritic process and even immunomodulatory. A natural inhibitor of cysteine proteases is cystatin C. METHODS: Cystatin C-deficient, sufficient and heterozygous mice were tested for onset, incidence and severity of CIA. The effect of cystatin C-deficiency was further dissected by testing the inflammatory effector phase of CIA; that is, collagen antibody-induced arthritis model and priming phase, that is, T cell response both in vivo and in vitro. In addition, in order to determine the importance of T cells and antigen-presenting cells (APCs), these cell populations were separated and in vitro T cell responses determined in a mixed co-culture system. Finally, flow cytometry was used in order to further characterize cell populations in cystatin C-deficient mice. RESULTS: Here, we show that mice lacking cystatin C, develop arthritis at a higher incidence and an earlier onset than wild-type controls. Interestingly, when the inflammatory phase of CIA was examined independently from immune priming then cystatin C-deficiency did not enhance the arthritis profile. However, in line with the enhanced CIA, there was an increased T cell and B cell response as delayed-type hypersensitivity reaction and anti-CII antibody titers were elevated in the cystatin C-deficient mice after immunization. In addition, the ex vivo naive APCs from cystatin C-deficient mice had a greater capacity to stimulate T cells. Interestingly, dendritic cells had a more activated phenotype in naive cystatin C-deficient mice. CONCLUSIONS: The lack of cystatin C enhances CIA and primarily affects in vivo priming of the immune system. Although the mechanism of this is still unknown, we show evidence for a more activated APC compartment, which would elevate the autoimmune response towards CII, thus resulting in an enhanced development of chronic arthritis.
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| 18. |
- Crandall, H, et al.
(författare)
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Bb2Bb3 regulation of murine Lyme arthritis is distinct from Ncf1 and independent of the phagocyte nicotinamide adenine dinucleotide phosphate oxidase
- 2005
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Ingår i: American Journal of Pathology. - 1525-2191. ; 167:3, s. 775-785
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Tidskriftsartikel (refereegranskat)abstract
- Several quantitative trait loci regulating murine Lyme arthritis severity have been mapped, including a highly significant linkage found on chromosome 5, termed Bb2Bb3. Within this region, the Ncf1 gene of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has recently been identified as a major regulator of arthritis severity in rodent models of rheumatoid arthritis, an effect attributed to protective properties of reactive oxygen species. To assess the role of Ncf1 in Lyme arthritis, we introgressed Bb2Bb3 from severely arthritic C3H/He mice onto mildly arthritic C57BL/6 mice. This increased Lyme arthritis severity, whereas the reciprocal transfer conferred protection from disease. A single nucleotide polymorphism was identified in the Ncf1 gene that did not influence the protein sequence or expression of Ncf1. Although polymorphonuclear leukocytes from C57BL/6 mice generated a greater oxidative burst than polymorphonuclear leukocytes from C3H/He mice, studies with the Bb2Bb3 congenic mice demonstrated this difference was not linked to Ncf1 alleles. Furthermore, Lyme arthritis severity was not altered in mice lacking either the Ncf1 or Gp91phox subunits of the NADPH oxidase complex. Together, these results argue that Ncf1 is not a candidate gene for regulation of Lyme arthritis and reveal Lyme arthritis to be independent of NADPH oxidase activity, distinguishing it from other models of rheumatoid arthritis.
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| 22. |
- Ge, C. R., et al.
(författare)
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Antibodies to Cartilage Oligomeric Matrix Protein Are Pathogenic in Mice and May Be Clinically Relevant in Rheumatoid Arthritis
- 2022
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Ingår i: Arthritis & Rheumatology. - : Wiley. - 2326-5191 .- 2326-5205.
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Tidskriftsartikel (refereegranskat)abstract
- Objective Cartilage oligomeric matrix protein (COMP) is an autoantigen in rheumatoid arthritis (RA) and experimental models of arthritis. This study was undertaken to investigate the structure, function, and relevance of anti-COMP antibodies. Methods We investigated the pathogenicity of monoclonal anti-COMP antibodies in mice using passive transfer experiments, and we explored the interaction of anti-COMP antibodies with cartilage using immunohistochemical staining. The interaction of the monoclonal antibody 15A11 in complex with its specific COMP epitope P6 was determined by x-ray crystallography. An enzyme-linked immunosorbent assay and a surface plasma resonance technique were used to study the modulation of calcium ion binding to 15A11. The clinical relevance and value of serum IgG specific to the COMP P6 epitope and its citrullinated variants were evaluated in a large Swedish cohort of RA patients. Results The murine monoclonal anti-COMP antibody 15A11 induced arthritis in naive mice. The crystal structure of the 15A11-P6 complex explained how the antibody could bind to COMP, which can be modulated by calcium ions. Moreover, serum IgG specific to the COMP P6 peptide and its citrullinated variants was detectable at significantly higher levels in RA patients compared to healthy controls and correlated with a higher disease activity score. Conclusion Our findings provide the structural basis for binding a pathogenic anti-COMP antibody to cartilage. The recognized epitope can be citrullinated, and levels of antibodies to this epitope are elevated in RA patients and correlate with higher disease activity, implicating a pathogenic role of anti-COMP antibodies in a subset of RA patients.
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| 23. |
- Geng, H., et al.
(författare)
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Incomplete B cell tolerance to cartilage oligomeric matrix protein in mice
- 2013
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Ingår i: Arthritis and Rheumatism. - : Wiley. - 0004-3591 .- 1529-0131. ; 65:9, s. 2301-2309
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: Cartilage oligomeric matrix protein (COMP) is a major noncollagenous component of cartilage and is used as a biomarker in rheumatoid arthritis and experimental arthritis. Injection of COMP leads to severe inflammatory joint disease, and antibodies play a critical role in mediating arthritis. The arthritogenicity of COMP might be due to the lack of self tolerance. This study was undertaken to determine the status of COMP-specific B cell tolerance using COMP-deficient mice. METHODS: Arthritis development and antibody responses were compared between COMP-sufficient and COMP-deficient littermates after immunization with rat COMP. Serum anti-COMP antibody levels were measured using a panel of recombinant mouse COMP proteins, and antibody-secreting cells were enumerated by enzyme-linked immunospot assays. A novel sandwich enzyme-linked immunosorbent assay was developed to assess COMP molecules in serum. RESULTS: COMP-sufficient mice, but not COMP-deficient mice, developed severe arthritis following immunization with rat COMP. However, anti-COMP antibody titers to native COMP and recombinant protein domains covering the entire mouse COMP sequence, except the less immunodominant type 3 repeat domains, were decreased in COMP-sufficient mice compared to COMP-deficient mice. In addition, COMP-sufficient mice had fewer B cells secreting COMP-reactive antibodies. Detectable levels of full-length COMP in arthritic COMP-sufficient B10.Q NCF-1(*/*) and healthy mice suggested systemic availability of COMP to the immune system. CONCLUSION: The lack of arthritis, together with high levels of COMP-specific antibodies, in COMP-deficient mice indicates that susceptibility to arthritis is COMP specific and that endogenous expression of COMP in wild-type mice tolerizes B cells in vivo.
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| 24. |
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| 25. |
- Grimm, Melissa J, et al.
(författare)
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Monocyte- and macrophage-targeted NADPH oxidase mediates antifungal host defense and regulation of acute inflammation in mice
- 2013
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Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 190:8, s. 4175-4184
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Tidskriftsartikel (refereegranskat)abstract
- Chronic granulomatous disease, an inherited disorder of the NADPH oxidase in which phagocytes are defective in the generation of superoxide anion and downstream reactive oxidant species, is characterized by severe bacterial and fungal infections and excessive inflammation. Although NADPH oxidase isoforms exist in several lineages, reactive oxidant generation is greatest in neutrophils, where NADPH oxidase has been deemed vital for pathogen killing. In contrast, the function and importance of NADPH oxidase in macrophages are less clear. Therefore, we evaluated susceptibility to pulmonary aspergillosis in globally NADPH oxidase-deficient mice versus transgenic mice with monocyte/macrophage-targeted NADPH oxidase activity. We found that the lethal inoculum was >100-fold greater in transgenic versus globally NADPH oxidase-deficient mice. Consistent with these in vivo results, NADPH oxidase in mouse alveolar macrophages limited germination of phagocytosed Aspergillus fumigatus spores. Finally, globally NADPH oxidase-deficient mice developed exuberant neutrophilic lung inflammation and proinflammatory cytokine responses to zymosan, a fungal cell wall-derived product composed principally of particulate beta-glucans, whereas inflammation in transgenic and wild-type mice was mild and transient. Taken together, our studies identify a central role for monocyte/macrophage NADPH oxidase in controlling fungal infection and in limiting acute lung inflammation. The Journal of Immunology, 2013, 190: 4175-4184.
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| 33. |
- Imai, Y., et al.
(författare)
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Identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury
- 2008
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Ingår i: Cell. - : Elsevier BV. - 1097-4172 .- 0092-8674. ; 133:2, s. 235-249
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Tidskriftsartikel (refereegranskat)abstract
- Multiple lung pathogens such as chemical agents, H5N1 avian flu, or SARS cause high lethality due to acute respiratory distress syndrome. Here we report that Toll-like receptor 4 (TLR4) mutant mice display natural resistance to acid-induced acute lung injury (ALI). We show that TLR4-TRIF-TRAF6 signaling is a key disease pathway that controls the severity of ALI. The oxidized phospholipid (OxPL) OxPAPC was identified to induce lung injury and cytokine production by lung macrophages via TLR4-TRIF. We observed OxPL production in the lungs of humans and animals infected with SARS, Anthrax, or H5N1. Pulmonary challenge with an inactivated H5N1 avian influenza virus rapidly induces ALI and OxPL formation in mice. Loss of TLR4 or TRIF expression protects mice from H5N1-induced ALI. Moreover, deletion of ncf1, which controls ROS production, improves the severity of H5N1-mediated ALI. Our data identify oxidative stress and innate immunity as key lung injury pathways that control the severity of ALI.
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| 39. |
- Lahore, G. F., et al.
(författare)
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Vitamin D3 receptor polymorphisms regulate T cells and T cell-dependent inflammatory diseases
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 117:40, s. 24986-24997
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Tidskriftsartikel (refereegranskat)abstract
- It has proven difficult to identify the underlying genes in complex autoimmune diseases. Here, we use forward genetics to identify polymorphisms in the vitamin D receptor gene (Vdr) promoter, controlling Vdr expression and T cell activation. We isolated these polymorphisms in a congenic mouse line, allowing us to study the immunomodulatory properties of VDR in a physiological context. Congenic mice overexpressed VDR selectively in T cells, and thus did not suffer from calcemic effects. VDR overexpression resulted in an enhanced antigen-specific T cell response and more severe autoimmune phenotypes. In contrast, vitamin D3-deficiency inhibited T cell responses and protected mice from developing autoimmune arthritis. Our observations are likely translatable to humans, as Vdr is overexpressed in rheumatic joints. Genetic control of VDR availability codetermines the proinflammatory behavior of T cells, suggesting that increased presence of VDR at the site of inflammation might limit the antiinflammatory properties of its ligand.
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| 40. |
- Li, T. T., et al.
(författare)
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Pathogenic antibody response to glucose-6-phosphate isomerase targets a modified epitope uniquely exposed on joint cartilage
- 2023
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Ingår i: Annals of the Rheumatic Diseases. - : BMJ. - 0003-4967 .- 1468-2060. ; 82:6, s. 799-808
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Tidskriftsartikel (refereegranskat)abstract
- ObjectivesTo identify the arthritogenic B cell epitopes of glucose-6-phosphate isomerase (GPI) and their association with rheumatoid arthritis (RA). MethodsIgG response towards a library of GPI peptides in patients with early RA, pre-symptomatic individuals and population controls, as well as in mice, were tested by bead-based multiplex immunoassays and ELISA. Monoclonal IgG were generated, and the binding specificity and affinity were determined by ELISA, gel size exclusion chromatography, surface plasma resonance and X-ray crystallography. Arthritogenicity was investigated by passive transfer experiments. Antigen-specific B cells were identified by peptide tetramer staining. ResultsPeptide GPI(293-307) was the dominant B cell epitope in K/BxN and GPI-immunised mice. We could detect B cells and low levels of IgM antibodies binding the GPI(293-307) epitopes, and high affinity anti-GPI(293-307) IgG antibodies already 7 days after GPI immunisation, immediately before arthritis onset. Transfer of anti-GPI(293-307) IgG antibodies induced arthritis in mice. Moreover, anti-GPI(293-307) IgG antibodies were more frequent in individuals prior to RA onset (19%) than in controls (7.5%). GPI(293-307)-specific antibodies were associated with radiographic joint damage. Crystal structures of the Fab-peptide complex revealed that this epitope is not exposed in native GPI but requires conformational change of the protein in inflamed joint for effective recognition by anti-GPI(293-307) antibodies. ConclusionsWe have identified the major pathogenic B cell epitope of the RA-associated autoantigen GPI, at position 293-307, exposed only on structurally modified GPI on the cartilage surface. B cells to this neo-epitope escape tolerance and could potentially play a role in the pathogenesis of RA.
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