Group A Streptococcus (GAS) is a globally disseminated pathogen that causes >500,000 deaths yearly and is ranked as ninth leading infectious cause of human mortality by the World Health Organisation. The spectrum of disease ranges from superficial infections of the skin and epithelium to invasive and systemic infections. Although the interaction of GAS with neutrophils has been extensively studied much remains to be discovered about the role of macrophages, which are the first line of defence encountered by invading pathogens. In this study, the aim was to establish a means of deriving macrophages from primary monocytes and to study both the efficiency of macrophage killing of GAS and the macrophage cell death response to GAS infection. Here, we report that monocyte-derived macrophages are able to take up and kill GAS during in vitro infection. Production of reactive oxygen species by macrophages was elicited during infection, but not nearly in as high amounts as produced by neutrophils. Investigating the type of cell death induced by GAS, markers for both apoptosis and necroptosis can be found after 8 hours of infection. These results highlight that macrophages indeed are participating in the clearance of GAS and more studies are needed to understand the roles of macrophages in early control of GAS infection.

The aim of cancer immunotherapy is to eradicate tumours by inducing a tumour-specific immune response. This thesis focuses on how antibodies and peptides can improve antigen presentation and the subsequent tumour-specific T cell response. Tumour recognition by the immune system can be promoted through delivery of antigen in the form of a vaccine. One example is the development of a therapeutic peptide vaccine containing both CD4+ and CD8+ T cell epitopes. So far, peptide vaccinations have shown limited success in clinical trials and further improvements are needed, such as choice of adjuvant and T cell epitopes, as well as targeted delivery of peptides and adjuvants to the same DC. In paper I, we describe the development of a peptide-peptide conjugate (with a tumour T cell epitope) that, via immune complex formation and FcγR binding, enhance antigen uptake and activation of DCs. The conjugate consists of three tetanus toxin-derived linear B cell epitopes (MTTE) that were identified based on specific IgG antibodies in human serum. Three MTTE peptide sequences were conjugated to a synthetic long peptide (SLP) that consists of a T cell epitope derived from the desired target tumour. In paper II, the conjugate was evaluated in a modified Chandler loop model containing human blood, mimicking blood in circulation. The conjugate was internalised by human monocytes in an antibody-dependent manner. A conjugate containing the model CMV-derived T cell epitope pp65NLV generated recall T cell responses dependent on MTTE-specific antibodies and the covalent conjugation of the three MTTE with the SLP. In paper III, a CD40-specific antibody was characterised for local treatment of solid tumours. The antibody eradicated bladder tumours in mice and induced T cell-mediated immunological memory against the tumour. In paper IV, we characterised the Chandler loop model (used in paper II) for its potential use in predicting cytokine release syndrome (CRS) in response to monoclonal antibodies (mAbs). Superagonistic antibodies (e.g., OKT3) induced rapid cytokine release whereas no cytokine release was induced by antibodies (e.g., cetuximab) associated with low incidence of CRS in the clinic. In conclusion, this thesis work demonstrates proof-of-concept of improved strategies for antibody- and peptides-based cancer immunotherapies and their potential use in multiple cancer indications.

Systemic lupus erythematosus (SLE) is a systemic inflammatory disease. Clinically, the distinction between ongoing inflammation attributed to SLE, and organ damage due to medication or co-morbidities remains challenging. In addition, SLE is a heterogeneous disease where the various disease phenotypes complicate the search for biomarkers that adequately reflect disease activity and/or signs of increasing organ damage. The aim of the thesis was to investigate and evaluate potential new biomarkers of disease activity and/or organ damage in SLE patients. High mobility group box protein-1 (HMGB1) is a nuclear non-histone protein that can shuttle to the cytoplasm, become secreted extracellularly, and participate in systemic inflammation. Administration of monoclonal anti-HMGB1 antibodies has been reported both to attenuate and intensify disease in animal models of arthritis and lupus. In Paper I of the thesis, circulating anti-HMGB1 was found in 23% of the SLE patients and correlated with disease activity variables. The biological role of these autoantibodies remains to be elucidated. As a consequence of massive circulating levels of cellular debris and immune complexes, SLE patients have insufficient capacity to remove such material via the reticuloendothelial system. Pentraxin 3 (PTX3) may possibly protect against lupus flares due to classical complement activation, opsonization of apoptotic cells, and cytokine induction. In Paper II, circulating PTX3 was found to be inhibited or exhausted by interferon (IFN)-α, a key cytokine of SLE pathogenesis, and serum levels of PTX3 in SLE patients were inversely related to IFN-α levels. Suppressed PTX3 levels may contribute to a vicious circle resulting in impaired waste clearance, autoantigen exposure and autoantibody production, and sustained disease activity. Osteopontin (OPN), a protein known to influence cell signaling and apoptosis, has been proposed as a marker of organ damage in pediatric lupus. In a Swedish cross-sectional study, circulating OPN levels were found to be raised in SLE (Paper III). In patients with recent-onset disease, OPN reflected disease activity, while in established disease, OPN appeared to mirror damage accrual and cardiovascular damage. In Paper IV, OPN was instead analyzed in an international longitudinal multi-center study based on patients with recent-onset SLE and follow-up data. OPN turned out to be a poor predictor of organ damage, but significant associations were observed between OPN and disease activity both at disease onset, as well as over 5 years of follow-up. In conclusion, increased anti-HMGB1 antibody and decreased PTX3 levels could potentially sustain the impaired waste-disposal. Of the molecules analyzed in this thesis, OPN seems to be the best marker of disease activity. Further studies of these proteins may help to better understand SLE pathogenesis and to optimize treatment of patients.

Mast cells are key players of the innate immune system due to their location at the interface of host and pathogen encounters, such as on mucosal surfaces or the skin. Secreting a variety of compounds, they communicate with other immune cells in a highly specific manner. Subsequently, reinforcements against foreign invaders are recruited while also defending the host, using bacteriolytic effector molecules. One type of pathogens which are competent challengers of the host’s immune system are Streptococci, causing a burden for humans and animals. Streptococcus equi subspecies equi is one example, a highly contagious horse pathogen with a silent carrier subset, causing “strangles”, a disease resulting in equine morbidity and mortality all over the world. The present study aimed to explore the virulence factors in S. equi responsible for immune system activation, represented by mast cells. Knockout mutants of the genes aroB, hasA, pyrC, recA, sagA and a combination of those, including a deletion strain of all superantigens (seeHILM), were co-cultivated with murine bone-marrow-derived mast cells (BMMCs). Mast cells alone and S. equi strain 4047 (wild-type) were used as controls. It was shown that 4 h after encounter of the bacteria, BMMCs responded with IL-6, TNF-α and MCP-1 secretion, indicating an inflammatory response to all strains except against the sagA mutant (ΔsagA) or the multi-deletion strain, the latter lacking several virulence factors including sagA. These results were confirmed at the mRNA-level where IL-6, TNF-α and Nr4a3 gene expression was significantly upregulated in BMMCs after 4 h incubation with wild-type S. equi. In contrast, when BMMCs were co-cultivated with sagA-deficient S. equi, no detectable upregulation was seen. These results were further confirmed in peritoneal-derived mast cells. After 24 h no secretion of cytokines was detected in response to ΔsagA mutants, in contrast to the strong cytokine output in response to wild-type S. equi. To elucidate the role of SagA, the precursor of streptolysin S (SLS), lysis was determined, and it was observed that ΔsagA does not lyse mast cells in contrast to wild-type with intact SLS. Transwell-based experiments indicated a partially contact-dependent response of mast cells to bacteria. Taken together, this study shows for the first time that SLS is the major mast cell activator produced by S. equi. I suggest the possible mechanism of cell death by lysis and reprogrammed signaling pathways of the host by sublytic concentrations of SLS, resulting in damage associated pattern-mediated signaling as well as auto- and paracrine amplification by inflammatory cytokines and other messenger molecules.

Characteristics as unknown cause, complicated pathophysiology and a great amount of complexity are describing systemic lupus erythematosus (SLE) more than well. It’s an autoimmune disease that is almost exclusive for women in their reproductive years and are believed to correlate with both genetics and environmental factors. Risk factors like stress, usage of cigarettes or birth controls with estrogen and infections are believed to trigger the progression of SLE. The spectra of therapeutic drugs are narrow due to the complexity and requirement of financial resources for scientific causes. Treatment is mainly symptomatic. The alternative splicing (AS) is a highly complex mechanism that is essential and are able to generate a great diversity of proteins encoded by the same gene are referred to as isoforms. Splicing occurs after the transcription that generates pre-mRNA because the exons need to fuse together and excision of introns. In patients with cancer diagnosis, they have observed that progression of disease and AS are correlated by the means of isoforms and splicing regulators. In studies, the relevance of alternative splicing events in SLE has been shown for both splicing regulators like SRSF1 and different isoforms for example CD44 and CD45. The aim of this study was to evaluate the potential biomarker AS in SLE.  This study of literature started with looking for clinical trials within databases like PubMed and Web of Science, that matched the aim of the study. Usage of terms like ‘SLE and biomarker’ och ‘SLE and alternative splicing’ etcetera. After inclusion of six scientific articles the author started the work with this literature of study.  Results gave strong indications that usage of alternative splicing as biomarker do have strong potential. Although the need of more goal-oriented scientific studies is required. Results from all six studies can be summarized by the line of argument that AS, in different ways, are somehow involved in the pathogenesis and progression of SLE. Both spliceosome, isoforms, splicing factors, other proteins and is also a possible, in the future, therapeutic target for example monoclonal antibodies. Other therapeutic targets maybe against phosphatases and kinases. New strategies are going to bring hope for the patients that are suffering from SLE, especially when the disease is active for 2–3 years. Being able to individualize treatment are going to generate a better quality of life for many SLE patients and usage of AS as a biomarker for disease severity.

Bakgrund: Luftvägarna inkluderar flera olika delar bland annat näsa, bihålor, svalg, struphuvudet, luftstrupen och lungor. Luftvägarna kan infekteras av virus eller bakterier och kan leda till inflammation. De olika varianter av virus som kan orsaka luftvägsinfektion är rhinoviruset, influensavirus (RS-virus), coronavirus samt bakterier av streptokocker eller pneumokocker. En variant av coronavirus med namnet, SARS-CoV-2 har orsakat utbrott i världen och förklarades som pandemi 11 mars 2020. I genomsnitt brukar vuxna insjukna i luftvägsinfektion ca 2-4 gånger per år, medan barn insjuknar oftare, 6-8 gånger om året. Immunförsvaret består av komponenter som samverkar för att angripa bakterier, svamp samt virus och skydda kroppen mot infektioner. Vitamin-D har en väsentlig påverkan på immunförsvaret, både det medfödda och adaptiva. Kroppen kan få i sig Vitamin-D genom kost och via UV-strålningen från solen.Vitamin-D har påverkan på immunförsvaret som leder till uppreglering av immunförsvarets väsentliga komponenter samt bidrar till att bekämpa infektion och skada cellmembran hos mikrober. Syfte: att undersöka om d-vitamin har skyddande effekt mot luftvägsinfektion. Metod: Följande litteraturstudie utgår från fem vetenskapliga artiklar från databasen, PubMed. Följande sökord var ”vitamin-D”, ”covid-19” och ”respiratory infection”.Resultat: Samtliga fem studier påvisade att tillskott av vitamin-D minskade förekomst av luftvägsinfektion samt att sjukdomsförloppet förkortades. I studie 5 minskade sannolikheten för covid-19 patienter med tillskott av vitamin-D att placeras i intensivvården. Det fanns signifikant skillnad i dosering av vitamin-D i studie 2 som jämfördes med en högre dos och en rekommenderad dos. Slutsats: Samtliga studier påvisar att vitamin-D har en viss skyddande effekt mot luftvägsinfektioner samt hjälper förkorta sjukdomsförloppet genom att stärka immunförsvaret. Studiernas resultat har varit varierande utifrån dess olika syfte samt behandlings tillvägagångssätt, således förekommit svårigheter att dra konkreta slutsatser. Doseringarna tolererades bra och orsakade inte allvarliga biverkningar som kunde kopplas till behandlingen. Det krävs ytterligare studier för att faställa vitamin-D:s skyddande effekter på luftvägsinfektion samtidigt om det förekommer individuella skillnader i upptag av vitamin-D samt metabolism. / Background: The respiratory tract includes several different parts including; nose, sinuses, pharynx, larynx, trachea and lungs. The respiratory tract can be infected by viruses or bacteria, which can lead to inflammation. The respiratory infection can be caused by rhinovirus, influenza virus, coronavirus or bacteria, such as streptococci or pneumococci. A variant of the coronavirus with the name, SARS-CoV-2 caused outbreaks over the entire world and was declared as a pandemic on 11th March 2020. On average, adults get a respiratory infection about 2-4 times a year, while children get sick more often, approximately 6-8 times a year. The immune system consists of components that work together to attack bacteria, fungi and viruses to protect the body against infections. Vitamin D has a significant effect on the innate and adaptive immune system. The body can absorb vitamin D through diet and from UV-radiation from the sun. Vitamin D has an effect on the immune system, which leads to upregulation of the immune system´s essential components and helps to fight infections as well as damaging cell membrane of microbes.  Objective: The purpose of the study was to investigate whether vitamin D has a protective effect in the fight against respiratory infections.  Method: The following literature study was based on five scientific articles from the databasePubMed. The following keywords were “vitamin d” and “respiratory infection”.Results: All five studies showed that vitamin D supplementation reduced the incidence of respiratory infections and the course of the disease was shortened. In study 5, the probability of covid-19 patients with vitamin D supplementation to be placed in intensive care decreased. There was a significant difference in the dosage of vitamin D in study 2, where higher dose was compared to a recommended dose.  Conclusion: All studies show that vitamin D has a certain protective effect against respiratory infections and helps shorten the course of the disease by strengthening the immune system. The results of the studies have varied depending on different aims and treatment approaches. Therefore it has been difficult to draw concrete conclusions. The dosages where well tolerated and didn’t cause serious side effects that could be linked to the treatment. Further studies are needed to determine the protective effects of vitamin D on respiratory tract infections and also toinvestigate whether there are individual differences in vitamin D uptake and metabolismthereby being able to draw concrete and general conclusions.

In this thesis, we have studied the immunological processes of joint inflammation that may be targets for future treatment of patients with arthritis. We focus on the immune-modulating properties of interferon-α (IFN-α) and uridine in experimental arthritis. The nucleoside uridine, which is regarded a safe treatment has anti-inflammatory properties notably by inhibiting tumor necrosis factor (TNF) release. Because the inflamed synovium in rheumatoid arthritis (RA) is characterised by pathogenic TNF-production, uridine could potentially be away to ameliorate arthritis. Systemic administration of uridine had no effect on antigeninduced arthritis (AIA), which is a T-cell dependent model where animals are immunized twice (sensitization) with bovine serum albumin (mBSA), before local triggering of arthritis by intra-articular antigen (mBSA) re-challenge. In contrast, intra-articular administration of uridine clearly down modulated development of AIA in a dose dependent manner and inhibited the expression of synovial adhesion molecules, influx of inflammatory leukocytes and synovial expression of TNF and interleukin 6, but did not affect systemic levels of proinflammatory cytokines or antigen-specific T-cell responses. Local administration of uridine may thus be a viable therapeutic option for treatment of arthritis in the future. Viral double-stranded deoxyribonucleic acid (dsRNA), a common nucleic acid found in most viruses, can be found in the joints of RA patients and local deposition of such viral dsRNA induces arthritis by activating IFN-α. Here we show that arthritis induced by dsRNA can be mediated by IFN-producing dendritic cells in the joint and this may thus explain why viral infections are sometimes associated with arthritis. Earlier, to study the effect of dsRNA and IFN-α in an arthritis model, that like RA, is dependent on adaptive immunity, dsRNA and IFN-α were administered individually during the development of AIA. Both molecules clearly protected against AIA in a type I IFN receptor-dependent manner but were only effective if administered in the sensitization phase of AIA. Here we show that the anti-inflammatory effect of IFN-α is critically dependent on signalling via transforming growth factor β (TGF-β) and the enzymatic activity of indoleamine 2,3 dioxygenase 1 (IDO). The IDO enzyme is produced by plasmacytoid DC and this cell type was critically required both during antigen sensitization and in the arthritis phase of AIA for the protective effect of IFN-α against AIA. In contrast, TGF-β and the enzymatic activity of IDO were only required during sensitization, which indicate that they are involved in initial steps of tolerogenic antigen sensitization. In this scenario, IFN- α first activates the enzymatic activity of IDO in pDC, which converts Tryptophan to Kynurenine, which thereafter activates TGF-β. Common for IDO-expressing pDC, Kyn and TGF-β is their ability to induce development of regulatory T cells (Tregs). We found that Tregs were crucial for IFN-α-mediated protection against AIA, but only in the arthritis phase. In line with this, adoptive transfer of Tregs isolated from IFN-α treated mice to recipient animals in the arthritis phase clearly protected against AIA. The numbers of Tregs were not significantly altered by IFN-α but IFN-α increased the suppressive capacity of Tregs against antigen-induced proliferation. This enhanced suppressive activity of Tregs in the arthritis phase was dependent on the earlier activated enzyme IDO1 during the sensitization phase of AIA. Thus, presence of IFN-α at the time of antigen sensitization activates the enzymatic activity of IDO, which generates Tregs with enhanced suppressive capacity that upon antigen re-challenge prevents inflammation. We have thus identified one example of how immune tolerance can be developed, that may be a future way to combat autoimmunity.

Antibodies passively administered together with their specific antigen can enhance or suppress the specific antibody response. This phenomenon is known as antibody feedback regulation. Whether this modulation causes up- or downregulation of the antibody response depends both on the antibody isotype and the antigen used. IgG antibodies passively administered together with particulate antigens, e.g. erythrocytes, can completely prevent the induction of an antibody response to the antigen. The suppressive capacity of IgG has been routinely used in the clinic since the 1960’s in RhD-prophylaxis to prevent hemolytic disease of the fetus and newborn. Although studied for decades, the underlying mechanism of IgG-suppression has remained elusive. The main focus of this thesis has been to elucidate the mechanism behind IgG-suppression of antibody responses in vivo in mouse models using intravenous immunization with specific IgG together with native or haptenated sheep red blood cells, SRBC. We show that IgG-suppression of IgM and long-term serum IgG-responses operates independently of activating FcγRI, III, IV, or the inhibitory FcγRIIB, thus confirming and extending previous findings. Moreover, we demonstrate for the first time that C1q, C3 and CR1/2 are dispensable for IgG-suppression of antibody responses. These findings strongly argue against the involvement of Fc-dependent mechanisms as the explanation for IgG-suppression. Interestingly, GC formation occurs in IgG-suppressed mice although the antibody response to surface SRBC epitopes are completely suppressed. The data suggests that these GCs develop in response to intracellular SRBC epitopes as well as to the passively administered suppressive IgG. Moreover, we demonstrate that passively administered IgG suppresses several parameters of an antibody/B cell response including antigen specific GC and non-GC B cells, extra-follicular antibody secreting cells, long-lived plasma cells and induction of immunological memory. Before the onset of the present study, two mechanisms appeared compatible with the majority of experimental findings: IgG-mediated antigen clearance and epitope masking. Herein we show that the contribution of IgG-mediated antigen clearance is negligible and that suppression of IgG-responses is strictly epitope specific. This provides compelling evidence that a very important mechanism underlying IgG-suppression is epitope masking.

An intact complement system is essential for the initiation of a normal antibody response. Antibodies can regulate their own production against the antigens that they are specific for. Both IgG3 and IgM are able to enhance the antibody response via complement. Here, we have compared the fate of OVA-TNP (ovalbumin-2,4,6-trinitrophenyl) administered intravenously to mice either alone or in complex with monoclonal IgG3 anti-TNP. IgG3-antigen complexes bind to marginal zone (MZ) B cells via complement receptors 1 and 2 (CR1/2) and are transported into splenic follicles. The majority (50% - 90%) of the antigens is deposited on follicular dendritic cells (FDC) and the antigen distribution pattern is strikingly similar to peripheral dendrites/processes of FDC already 2 h after immunization. The development of germinal centers (GC) induced by IgG3-antigen complexes is impaired in mice lacking CR1/2. Experiments on bone marrow chimeric mice show that CR1/2 expression on both MZ B cells and FDC is required for optimal IgG3-mediated enhancement of antibody responses. Complement factors C3 and C1q are essential for OVA-TNP delivery and deposition on splenic FDC. The production of IgG anti-OVA is abrogated in mice lacking CR1/2, C1q, and C3. Further, IgG3-antigen complexes dramatically upregulate the memory response against OVA-TNP by inducing OVA-specific memory cells. Besides small protein OVA, IgG3 can also upregulate humoral responses against large soluble keyhole limpet hemocyanin. To further study the role of MZ B-cells and CR1/2 in enhancement of antibody responses, a knock-in mouse strain, Cμ13, was used. IgM in this mouse strain is unable to activate complement due to a point mutation in the constant µ-heavy chain. Cμ13 mice have a higher proportion of MZ B cells, with higher CR1/2 expression, than wild-type mice. More IgG3-immune complexes are captured by MZ B cells and deposited on FDC in Cμ13 than in WT mice. In spite of this, IgG3 did not enhance the primary antibody response more efficiently in Cμ13 mice. The existence of endogenous IgM-mediated feedback regulation was suggested by the observation that GC development and antibody responses, after priming and boosting with suboptimal doses of SRBC, was lower in Cμ13 than in WT mice.

Bakgrund: Att definiera matöverkänslighet är ännu idag inte fastställt, det råder delade meningar om denna definition. Författarna har valt att inrikta sig på matöverkänslighet som definieras matallergi och matintolerans. Flera av matallergierna kan utlösa en allergisk chock, denna typ av chock som även kallas anafylaktisk chock kan vara livshotande om inte behandling sätts in direkt. Idag lider var tredje person i Sverige av någon typ av allergi, vanligt förekommande matöverkänsligheter är komjölksallergi, laktosintolerans, äggallergi och nötallergi. Syfte: Syftet är att undersöka hur många människor i åldern 18-28 år som lider utav matöverkänslighet och om allergimedicin bärs med dagligen. Metod: En empirisk enkätstudie låg till grund för studiens resultat. Författarna valde att göra en kvantitativ studie för att kunna samla in data systematiskt och sedan sammanfatta dessa i statistisk form. Studien var ute efter ett större antal deltagare utspridda på olika platser så därför passade kvantitativ metod in med en enkätstudie. Det blev en prospektiv tvärsnittsstudie då studien undersöker hur något såg ut vid ett specifikt tillfälle. Resultat: Studiens resultat visar att drygt en tredjedel utav deltagarna lider utav någon form utav matöverkänslighet, allergi eller intolerans. Ytterligare 20 % har någon gång reagerat på ett livsmedel, men använder idag ingen medicin mot sina besvär. De vanligaste livsmedlen att reagera emot är mjölk- laktos, frukt, nötter och vete. Slutsatser: Knappt hälften av de personerna som deltagit i denna studie har någon gång reagerat på ett livsmedel. Sedan har drygt 35 % utav deltagarna uppgett att de använder någon form utav allergimedicin mot livsmedel. Det innebär att människor i västvärlden är mer matöverkänsliga nu än någonsin och trenden fortsätter att eskalera. Renlighet och matvanor är troligen orsaker till detta växande hälsoproblem.