New efficient vaccines against infectious diseases are in demand. Some important factors impeding the vaccine development are the poor immunogenicity and the MHC restriction of the immune responses to a number of antigens. The use of novel vaccine adjuvants or carrier proteins, which are known to enhance the immunogenicity of the subunit antigens and provide T-cell help, can circumvent these problems. The potential of heat shock proteins (HSPs) to function as adjuvants when fused to or co-delivered with protein antigens, make them attractive vaccine candidates. In this thesis we have evaluated the potency of heat shock protein 70 (HSP70) as a possible vaccine adjuvant and studied the mechanisms behind the adjuvanticity. The first article aims to evaluate the carrier effect of glutathione-S-transferase (GST) on a malarial antigen EB200 that induces a MHC restricted response in mice. Immunization of CBA and C57BL/6 mice, high and low responders to EB200, respectively, with the GST-EB200 fusion protein elicited EB200 specific antibody responses in both strains of mice, which indicated that MHC restriction was broken in C57BL/6 mice. However, the antibody affinity and the magnitude of the response were lower in the C57BL/6 mice compared with that in CBA. To improve the response, the efficacy of various adjuvants like alum, HSP70 from Trypanosoma cruzi, and the adjuvant combination (HSP70 and cholera toxin) was evaluated. The results indicated that cholera toxin and HSP70 act synergistically and improve the immunogenicity of EB200 antigen by increasing the affinity and magnitude of the response. HSP belongs to a family of conserved molecules and the maximum homology lies on the N-terminal region of the protein, therefore there is a risk that use of a complete molecule would give rise to autoimmunity. Thus, in our second study we first evaluated the adjuvant effect of the less conserved portion of HSP70 derived from Plasmodium falciparum (Pf70C). We found that the Pf70C exhibited similar adjuvant properties as the whole molecule. We further analyzed the adjuvant potential of Pf70C against EB200 formulated as a chimeric DNA vaccine construct. These constructs alone failed to generate substantial levels of EB200 specific antibodies in mice. However, the DNA immunization efficiently primed the immune system. This was evident as the subsequent boosting with the corresponding recombinant fusion proteins Pf70C-EB200 elicited strong EB200 specific Th-1 antibody responses. In contrast, no such priming effect was observed for ex vivo IFN-γ production, however stimulation with the Pf70C-EB200 fusion protein induced an enhanced secretion of IFN-γ in vitro. During the infection process, the synthesis of bacterial HSP is up-regulated, which is known to sensitize T cells in the infected host. Since a high degree of homology exists within the phylogenetic families of HSPs, we postulated that exposure of mice to microorganisms could prime the immune system for evolutionary diverse HSPs and for any antigen coupled to them. We tested this hypothesis by priming mice with different microorganisms such as BCG, Mycobacterium vaccae or Chlamydia pneumoniae and boosted with a recombinant fusion protein Pf70C-EB200 or with a panel of HSPs. We found that BCG and M. vaccae but not C. pneumoniae could provide priming of the immune system to induce secondary IgG responses to Pf70C as well as to other HSPs tested. The priming effect was also observed when the EB200 antigen was coupled to Pf70C. Analysis of the IgG1 and IgG2a profiles and IFN-g production induced against the HSPs revealed a mixture of Th1/Th2 type of responses. We also observed that HSP70 specific sera cross-reacted some extent with certain autoreactive antigens. However, no deposits were observed in the kidneys of HSP treated animals. Finally, we investigated the role of TLR2 and TLR4 on HSP70-mediated adjuvanticity. We found that HSPs displayed different degrees of adjuvanticity regarding both the strength and the profile of the induced immune response. Also, they possessed different requirements for signaling through TLRs. While HSP70 from T. cruzi induced antigen-specific humoral responses in wild type as well as in both the TLR2 and TLR4 knockout mice, the response was diminished in the TLR4 knockout mice when both the whole and C-terminal fragment of HSP70 from Mycobacterium tuberculosis was used. However, the C-terminal fragment of P. falciparum HSP70 elicited responses only in wild type mice but not in TLR2 or TLR4 knockout mice indicating that the adjuvant function differ for phylogenetically related HSPs. Taken together our data suggest that HSPs can be promising candidates in future vaccines.

Coeliac disease (CD) is a multifactorial disease of the small intestine. In genetically predisposed individuals the, ingestion of cereals leads to a remodulation of the mucosal architecture, and the production of autoantibodies against tissue transglutaminase (tTG). The treatment is a lifelong gluten-free diet. The diagnostic procedure relies on the examination of a small-bowel biopsy that displays villous atrophy. A spectrum of clinical manifestations is associated with CD, ranging from overt enteropathy to atypical and silent symptoms. Approximately 1% of the general population has CD, and the majority is undiagnosed. Although most patients with active CD can be detected by the assessment of elevated IgA-tTG, some patients lack these antibodies. Moreover, individuals with IgA-deficiency cannot be identified by means of IgA serology. The aim of this thesis was to investigate the clinical utility of IgG-tTG for the detection and follow-up of subjects with active CD. The included studies showed that IgG-tTG was highly prevalent in IgA-deficient and IgA-competent patients with CD, whereas non-CD patients rarely had these antibodies. During a gluten-free diet, IgG-tTG decreased, demonstrating that IgG-tTG can be used to follow the patient’s adherence such a diet. Furthermore, 10% of healthy IgA deficient blood donors had elevated IgG-tTG, indicating that they had silent CD. In IgA-competent subjects, high IgG-tTG levels correlated with a severe mode of CD and profound mucosal deterioration, suggesting that IgG-tTG might be involved in the disease progression. Moreover, we found that although a considerable percentage of IgA-competent patients lack IgG-tTG, the presence of these antibodies in conjunction with high levels of IgA-tTG was highly predictive of a severe small-intestine villous atrophy. It was also demonstrated that IgG-tTG normalisation coincided with clinical remission in IgA-competent CD patients on a gluten-free diet.

Type 1 diabetes (T1D) is due to a selective loss of the insulin producing β-cells. However, the process responsible for this loss is still unknown. There is accumulating evidence that enteroviruses (EVs) are involved in T1D. In addition to direct virus-induced cytolysis, EVs could facilitate β-cell destruction by inducing inflammatory cytokines. Induction of such genes has previously been shown in EV-infected islets in vitro. Modulation of inflammatory mediators expressed in the islets could be a possible strategy to reduce β-cell destruction. In the first paper we screened uninfected isolated human islets for genes with the potential to induce or modulate an immune response. We found that several of the genes expressed in the islets encode proteins with a powerful biological activity, such as IL-1β, IL-8, MIP-2α, MCP-1 and MIF. This indicates that the islets themselves can express several triggers of inflammation, and if expressed in vivo these mediators would probably contribute to β-cell destruction. The vitamin B3 derivate, nicotinamide (NA), has been shown to modulate expression of factors important for coagulation and inflammatory responses. Addition of NA into isolated islet cultures resulted in a reduced expression of the pro-inflammatory chemokine MCP-1 and the coagulation activator tissue factor, suggesting that NA may have implications for both inflammatory responses and the pro-coagulant activity of islets. We successfully isolated EVs from three newly diagnosed T1D patients. All isolates showed tropism for human islets and β-cells in vitro and clearly affected islet function. We also found that EV infection induced islet secretion of the chemokines IP-10 and MCP-1and that this induction could be blocked or reduced by addition of NA to the culture medium. Interestingly, NA also reduced viral replication and virus-induced islet destruction. To conclude, this thesis provides new information about expression and modulation of inflammatory mediators in infected and uninfected human islets that could trigger inflammatory reactions leading to β-cell destruction. Moreover, it further strengthens the causal relationship between EV and T1D.

ABSTRACT The Swedish Human Proteome Resource (HPR) is a project about mapping of human genes and proteins. It aims to describe the function and distribution of all human genes and corresponding proteins, using in-house produced antibodies and tissue microarrays (TMA) for enzyme based immunohistochemistry. The mono-specific antibodies are used for immunostaining of human tissue. Specific predicted antigens named Protein Epitope Signature Tag (PrEST) are needed to produce mono-specific antibodies. PrEST are produced using recombinant technology from predicted genes and used as immunogens to produce (mono-specific) antibodies in rabbits. In this study, 84 mono-specific antibodies with known specificity to human proteins were used for immunohistochemical analysis of mouse tissues to determine the cross reactivity between mouse and human. For 6 of the 84 antibodies the results differed between mouse and human tissue. A comparison between the PrEST used with the mouse proteome using database search programs showed homologies that were less than 100% in these six cases. Thus, future studies on cross reactivity will focus on how to increase the accuracy in its prediction at the in silico stage of the experiment.

ABSTRACT Allergy to mustard has been reported for many years, in some cases as severe anaphylactic reactions. Recent studies imply that this allergy is increasing. Three major allergens have been isolated and characterised; Sin a 1 and Sin a 2 in yellow mustard (Sinapis alba), and Bra j 1 in oriental mustard (Brassica juncea). Yellow mustard and black mustard (Brassica nigra) are the most common species in Europe, whereas oriental mustard is more frequent outside Europe. Mustard plants belong to the Brassicaceae/Cruciferae family. Mustard is present as an ingredient in different foods, sauces and spices, often in small amounts. According to the European labelling directives, mustard and products thereof must always be declared. To monitor this regulation, methods need to be developed to detect mustard. Polyclonal antibodies, produced in rabbits, against yellow and black mustard were characterised with immunodiffusion, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and immunoblotting. Rocket-immunoelectrophoresis and enzyme-linked immunosorbent assay (ELISA) were developed for the detection and quantification of mustard protein. With indirect competitive ELISA a concentration of 156ng mustard protein per ml food extract was detected, which is more than enough to cover the lowest reported reactive doses.

This thesis aimed to the identification of immune biomarkers of mycobacterial infection for better diagnosis of tuberculosis (TB) and also focused on new vaccination strategies with a particular emphasis on the immune responses in the respiratory tract using murine models. Since the lung is the natural habitat for the M. tuberculosis, we reasoned that immune responses detected locally in the lungs would be good correlates of infection (Paper I). Likewise, immune responses induced in the respiratory tract following immunization would be more effective against mycobacterial infection. We showed that cytokines (IL-12, TNF, and IFN-γ) and cytokine receptors (sTNFR1 and sTNFR2) together with specific antibodies in the respiratory tract correlated better with the bacterial burden in the organs. In Paper II, we investigated the role of the BCG vaccination as a priming vaccine in a heterologous prime-boost immunization protocol. The results showed that the neonatal BCG vaccination primed the immune system for a relevant antigen and showed a generalized adjuvant effect. Using this immunization protocol, protective immune responses in the lungs were generated independently of the route used for the booster immunization. In Paper III, We showed that exposure to mycobacterial antigens during the gestational period led to antigen transportation from the mother to the fetus and this resulted in an early priming of the fetal immune system. Immunization with the same antigen during the postnatal life increased antigen-specific recall IFN-γ responses and protection against infection. We examined the role of innate immunity for the induction of acquired immune responses upon immunization with mycobacterial antigens using TLR2 deficient mice (Paper IV). Our data indicated that suboptimal innate immune responses in the TLR2-/- mice might compromise the induction of acquired immune responses. Overall, the current findings suggested that a better understanding of the mucosal immunity would be useful for the improvement of diagnostic procedures and the development of efficient vaccines against TB. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript

Multiple sclerosis (MS) is an autoimmune disorder of the central nervous system (CNS). In the current thesis, we have preformed an immunological investigation of patients with MS and developed an immunosuppressive cell therapy that could be beneficial for these patients. MS has been considered to be driven by T helper type1 (Th1) lymphocytes but new data indicate the involvement of Th17 responses. T cells from patients with MS that were evaluated for immunological status secreted both interferon-γ and interleukin-17 upon stimulation. However, T cells from patients with MS in remission, in contrast to relapse, had poor proliferative capacity suggesting that they are controlled and kept in anergy. T regulatory cells (Tregs) are important to maintain self-tolerance and the role of CD4+CD25+FoxP3+ Tregs in autoimmunity has been extensively investigated. We analyzed Tregs from patients with MS in relapse and remission by multicolor flow cytometry for the expression of CD3, CD4, IL2R (CD25), FoxP3 and the IL7R (CD127). Patients in relapse exhibited higher levels of FoxP3-positive Tregs lacking CD25 compared to healthy controls, indicating that Tregs might attempt to restrain immune activity during relapse. In the murine experimental autoimmune encephalomyelitis (EAE) model of MS, therapy with suppressive cells such as Tregs or mesenchymal stromal cells (MSCs) has proven beneficial. However, systemic administration of such cells may immunologically compromise the recipient and promote infections due to general immunosuppression. We hypothesized that suppressive cells can be equipped with a CNS-targeting receptor and be delivered intra-nasally to avoid systemic exposure. CD4+ T cells were modified with a lentiviral vector system to express a myelin oligodendrocyte (MOG)-targeting receptor in trans with the FoxP3 gene that drives Treg differentiation. Genetically engineered Tregs demonstrated suppressive capacity in vitro and localized to the brain and suppressed ongoing encephalomyelitis in vivo. Cured mice were rechallenged with an EAE-inducing inoculum but remained healthy. MSCs are a heterogeneous population of stromal cells residing in most connective tissues and have the capacity to suppress effector cells of the immune system. MSCs were engineered to express MOG-targeting receptors using lentiviral vectors. Genetically engineered MSCs retained their suppressive capacity in vitro and successfully targeted the brain upon intranasal delivery. Engineered MSCs cured mice from disease symptoms and these mice were resistant to further EAE challenge. Encephalitic T cells isolated from cured mice displayed an anergic profile while peripheral T cells were still responsive to stimuli. In conclusion, MS patients have peripheral CNS-reactive T cells of both Th1 and Th17 type that, while in remission, are kept in anergy. Also, MS patients in relapse exhibit increased levels of CD25 negative Tregs indicating an attempt to restrain immune activity. Finally, immunosuppressive cells can be genetically engineered to target CNS and efficiently suppress encephalomyelitis in an active EAE model upon intranasal delivery.

Immune complexes (ICs) are produced during normal immune responses and facilitate clearance of foreign antigens. ICs not efficiently cleared from the circulation can cause tissue damage. This might happen if ICs are formed with autoantibodies and autoantigens. Well described effects of ICs are neutralization of antigen, classical complement activation or FcR-mediated phagocytosis, whereas cytokine inducing effects of ICs in human clinical settings are less well described. I have investigated cytokine-inducing properties in vitro of ICs from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and cryoglobulinemia in association with lymphoproliferative diseases. Cryoglobulin (CG)-induced cytokine production varied with changes in temperature and ionic strength in parallel to CG precipitation. IgG CG-induced cytokine production was also mediated via FcγIIa on monocytes. Blockade of the complement system, resembling the in vivo situation of complement consumption in CG-associated diseases, increased IgG CG induced IL-10 and decreased TNF-α production. This represents hitherto not described mechanisms for CG-associated inflammation. ICs from SLE patients induced IL-10 and IL-6 production from PBMC cultures via FcγRIIa. Occurrence of anti-SSA autoantibodies and signs of in vivo complement activation contributed to increased levels of circulating ICs in SLE patients, corresponding to increased amounts of IC-induced IL-10 in vitro. This represents a possible vicious cycle that might perpetuate antibody dependent pathology in SLE, and put anti-SSA in a new pathological context. RF-associated ICs from RA joints and ICs formed with antibodies against collagen type II from RA serum induced pro-inflammatory cytokine production from monocytes via FcγRIIa, showing how specific autoantibodies might induce or perpetuate joint inflammation in RA. I have described how ICs can induce significant amounts of pathophysiologically important monocyte-derived cytokines in three major IC-dependent diseases. Blockade of FcγRIIa and suppression of monocytes/macrophages might be a means of reducing pathogenic IC-induced cytokine production in these diseases.

During pregnancy, the woman carries a fetus partly foreign to her immune system, because of the expression of paternal antigens. Despite this, the fetus is normally tolerated and not rejected, as is often the case with organs in allogeneic transplantations. Systemic changes in maternal blood occur during pregnancy but, perhaps of greater importance, are changes in tissues locally in the uterus. The pregnant uterine endometrium, the decidua, is infiltrated by large numbers of leukocytes, mainly natural killer (NK) cells but also macrophages and T lymphocytes. Further, various cytokines are known to be secreted at the fetomaternal interface. However, the functions of these cells and the cytokine networks are not fully understood. The aim of this thesis was to investigate the local immune balance in normal human pregnancy decidua, both in the early phase of pregnancy and at parturition. First trimester decidual mononuclear cells, NK cells and macrophages were all shown to secrete IFN-γ, IL-4 and IL-10, as detected by ELISPOT. The secretion was not mirrored in blood from the same subjects. A significantly larger number of decidual macrophages secreted IL-10 than did their blood counterparts, indicating potential regulatory functions of this cell type. Further examination of early pregnancy decidual macrophages by microarray revealed 120 genes being differentially regulated at the transcriptional level in decidual compared to blood monocytes/macrophages. Several genes were associated with alternative activation/M2 polarization of macrophages, including CCL-18, CD209, IGF-1, MRC-1 and FN-1. Genes connected to immune regulation and tissue remodelling were common, in line with the potential functions for this cell type in utero. In addition, some molecules not previously connected to decidual macrophages, such as TREM-2, A2M and PGDS, were found to be upregulated, gaining new insights into the regulatory functions of decidual macrophages. Term decidual mononuclear cells spontaneously secrete IFN-γ, TNF, IL-4, IL-10, and TGF-β. No differences were seen between tissues obtained before and after the onset of labour, indicating that decidual mononuclear cells are not the main cell population responsible for plausible cytokine regulation in the process of labour induction. Placental and fetal membranes as well as cells in the maternal systemic circulation may instead contribute to a possible shift in immune balance prior to pregnancy termination. In conclusion, decidual leukocytes, including NK cells and macrophages, are potential producers of both Th1-like/pro-inflammatory and Th2-like/anti-inflammatory cytokines in early pregnancy as well as at parturition. Decidual macrophages are of a specialized phenotype with effector functions contributing to a proper invasion of the placenta and to immunological protection of the semi-allogeneic fetus. This thesis adds new knowledge on local immune balance during normal human pregnancy, however, the clinical significance of the presented data needs to be clarified.

<p><em>Background </em>The proliferation signal inhibitors (PSIs) sirolimus (SRL) and everolimus (ERL) are the potent immunosuppressive drugs using in organ transplantation and has been used successfully in renal transplant recipients (RTX) as well. PSIs are the key factors to overcome the allograft rejections after successful organ transplantation since the immune system starts to react against the graft. SRL and ERL prevents the action of immune system b inhibits the proliferation of T- and B-cells by inhibiting the intracellular signaling of interleukin-2. The presence of excess amount of serum proteins including albumin in the urine is considered as proteinuria, which reflects the loss of kidney function. The occurrence of proteinuria can be the result of abnormal glomerular filtration and/or impaired tubular endocytic function of renal proximal tubular epithelial cells (PTECs). Megalin and cubulin are two scavenger receptors present on epical surface of PTECs and involved in reabsorption of proteins after glomerular ultrafiltration process in the kidney. Proteinuria appears too high in renal transplanted patients during ongoing   treatment with PSIs.</p><p><em>Aim</em> Our study aimed to investigate and correlate the expression level of megalin and cubilin and albumin uptake in PTEC of renal transplanted patients before and after conversion to PSI.</p><p><em>Methods</em> To retrieve the maximal expression of our interest molecules in renal PTECs, we optimized antigen retrieval (AR) method and primary antibody dilution for each molecule separately. An optimization experiment was performed on 3 different normal patients renal biopsies were used. Later, human renal biopsy specimens originated from 4 different renal transplanted patients were used in this study. From all the 4 patients biopsy specimens were taken before and ongoing administration of PSIs (SRL, ERL). The expression of megalin, cubilin and albumin uptake in PTEC of renal transplant patients was determined by immunohistochemical staining.</p><p><em>Results</em> Based on the optimization experiments, we selected the AR method and primary antibody dilution for the expression of megalin, cubilin and albumin uptake. In 4 renal transplanted patients following administration of PSIs results in patients 1, 2, 3 expression of megalin, cubilin and albumin uptake during ongoing PSI treatment was not comparable or even more intense than before PSIs introduction. The expression of megalin, cubilin and albumin uptake was reduced in patient 4 during ongoing PSI treatment.</p><p><em>Conclusion</em> Our findings suggest that the renal transplant patient 4 developed proteinuria during PSI medication. The expression of megalin, cubilin and albumin uptake was markedly decreased during ongoing PSI treatment in patient 4. We concluded that there is a direct link between PSI medication and tubular dysfunction, which might cause proteinuria</p>