Rheumatoid arthritis (RA) is an autoimmune disease with systemic inflammatory features that primarily affects small peripheral joints. Type II collagen (CII), is the most abundant collagen type in joint cartilage. Antibodies against CII (anti-CII) are found in a subpopulation of RA patients. Anti-CII can form surface-bound immune complexes (IC) in inflamed joints, which might intensify joint inflammation and destruction. In this thesis I have studied the functional effects of surface-bound anti-CII–containing IC in vitro and correlated the results to clinical parameters. Anti-CII IC induced TNF-α, IL-1β and IL-8 production from monocytes via FcγRIIa. Anti-CII levels were dichotomously distributed in RA patients where a small outlier group (3.3%) with very high anti-CII levels showed in vitro induction of pro-inflammatory cytokines by anti-CII-containing IC. These patients also had a distinct phenotype with elevated laboratory signs of inflammation and increased radiological erosions at the time of diagnosis. In another in vitro model, co-cultured macrophages and RA synovial fibroblasts stimulated with anti-CII IC induced the production of matrix metalloproteinases (MMP)-1 and MMP-8, enzymes responsible for the initial cleavage of CII during cartilage degradation. This was mediated via production of TNF-α and IL-1β, and especially anti-CII IC-induced IL-1β sup-ported the production of MMP-1. The presence of anti-CII antibodies in patients with early synovitis was not predictive for future RA development. In summary, I have shown how anti-CII-containing IC may explain part of the early pathogenesis and can define a distinct clinical phenotype in RA patients with high levels of anti-CII.

Immune complexes (IC) have key pathological roles in both autoimmune and infectious diseases. In this thesis functional mechanisms behind IC-driven inflammation in rheumatic diseases and tropical infections have been studied, with special focus on the contribution of autoantibodies and cytokine-inducing properties of IC. In the autoimmune disease SLE, increased levels of IC-induced cytokines were associated with both increased classical complement activation and the occurrence of the autoantibodies anti-SSA and anti-SSB, both directed against RNA-associated antigens. In addition, complement activation and anti-SSA synergistically predisposed to higher levels of IC in sera. In the following study it was demonstrated that also other autoantibodies against RNA-associated autoantigens were more enriched than anti-dsDNA in SLE IC. Sudanese Visceral Leishmaniasis (VL) patients had elevated IC levels, and precipitated IC induced higher levels of GM-CSF, IL10, IL6 and IL1RA than control IC. Levels of IC were especially prominent in severely ill patients receiving antimony treatment, and a parallel association with IC induction of GM-CSF was demonstrated. Leishmania-infected patients were often rheumatoid factor (RF) positive and a substantial number displayed reactivity towards cyclic citrullinated peptide (CCP) antigens. Contrary to what was seen in Sudanese RA sera, the CCP reactivity was not restricted to citrulline but reacted equally well with arginine-containing control peptides. Levels of anti-CCP among VL patients were not due to cross-reactions with, or CCP-reactivity bound to IC. I have demonstrated that IC are associated with the presence of autoantibodies in both SLE and in Leishmania infection. In SLE, autoantibodies against RNA-associated antigens were more prone to form circulating IC than anti-dsDNA. In Leishmania infection false reactivity against the CCP-autoantigen correlated to IC levels although the IC themselves did not contain such reactivity. In both diseases higher IC levels were associated with a more active disease, and purified IC induced key cytokines in disease pathogeneses.

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.

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>

<p>T lymphocytes can be rendered specific against a wide range of antigens by the genetic transfer of a chimeric receptor, a fusion between the antigen-binding domain of an antibody and the signalling domain of a T cell receptor. The use of such chimeric T lymphocytes has shown promising results for cancer therapy. Previous experiments in our laboratory have shown low rates of gene transfer using retroviral vectors. In this study, investigations have been done to increase the number of genetically modified cells. Different enhancers such as PLL and polybrene have previously been used in combination with retroviral transduction. The optimal retroviral protocol in this study showed to be the use of retrovectors produced with twice the normal concentration of the plasmids encoding env and gag-pol rather than the use of the enhancers. A 6-day pre stimulation of T lymphocytes prior transduction together with a centrifugation step increased the rate of modified cells even further. Alternative approaches of gene transfer were also investigated, including plasmid transfection and adenoviral transduction. While transfection protocols yielded low numbers of modified cells, adenoviral vectors showed the highest rate of gene transfer.</p> / <p>Cancer är den sjukdom som idag, efter hjärt-kärl-sjukdomar, kräver flest dödsfall i i-länder. Som en alternativ behandlingsmetod mot cancer pågår just nu forskning om genetiskt förbättrade immunceller, s.k. chimära T lymfocyter, skulle kunna användas för att döda tumörceller. De chimära cellerna är utrustade med en konstgjord receptor som är en fusion av en antikropp och en signalkedja. Det gör att cellerna kan riktas mot ett brett urval av cancertyper. Att få cellerna att ta upp generna som behövs för den konstgjorda receptorn har visats sig vara problematiskt. Den här studien har därför som mål att förbättra cellernas förmåga att ta upp gener. För detta har vi använt oss av retrovirus- och adenovirus-system tillsammans med försök att få cellerna att spontant ta upp generna, sk. plasmid-transfektion. Studien har visat att de båda virussystemen ger högst antal modifierade celler. Olika substanser som tidigare har visat sig förhöja graden av gentillförsel har testats, men vår studie har visat att tillverkningen av virusvektorerna har större påverkan på resultaten än någon av de olika hjälpmedlen.</p>

<p>The amyloidoses are biochemically heterogeneous diseases with patholophysiologic deposits of various proteins. Amyloid deposits can occur either localized to one organ or tissue or as part of a systemic disease with deposits in many different tissue. The clinical course, prognosis and therapy are different for each type of amyloidosis and therefore a type specific diagnosis is demanded as early as possible. We describe a method for typing of the most common systemic amyloidoses based on Western blot analysis combined with specific</p><p>in- house antibodies, using subcutaneous fat biopsies. We found that the method is reliable and easy to perform and the tissue sample needed is obtained by minor surgery.</p><p>In the aortic intima amyloid deposits are often associated with atherosclerosis plaques. In our study we also investigated the prevalence of intimal amyloid from 10 patients age 58-94, amyloid deposits were present in 50% of the cases.</p> / <p>Amyloidos är ett sjukdomstillstånd där proteiner som normalt är lösliga i kroppen felveckas och formar långa olösliga fibriller som ansamlas i vävnader och organ såsom t.ex. hjärta, hjärna och lever. Det finns cirka 25 proteiner som kan ge upphov till amyloidos. Man kan skilja på två huvudgrupper av amyloidos, systemisk och lokaliserad. Vid lokal amyloidos kan inlagringar förekomma i specifika vävnader vid framför allt vissa åldersberoende sjukdomar som t.ex. Alzheimers sjukdom. Vid systemisk amyloidos förekommer inlagringar i praktiskt taget alla vävnader. Symtomatologin vid systemisk amyloidos är variabel och sjukdomsbilden kan vara svårtolkad men tidig och specifik diagnostik ger möjlighet till riktad terapi mot den bakomliggande sjukdomen. Syftet med denna studie var att utvärdera en Western blot metod som använts för typning av vanligaste formerna av systemisk amyloidos. De slutsatser som nåtts är att denna metod är snabbt, pålitligt och enkel att utföra. Diagnos erhölls med finnålsbiopsi av bukfettvävnad som är enkel, snabb och billig metod med liten risk för patienternas hälsa. Vi lyckades också med hjälp av immunhistokemisk infärgning titta på prevalens av amyloid i aortas intima.</p>

The islets of Langerhans are small and delicate spheroid organs scattered in the pancreas responsible for insulin production. Transplantation of isolated islets is a beneficial therapy for patients with a severe form of type 1 diabetes. The islets, which normally are richly vascularized in the pancreas, are completely disconnected from the vascular support by the enzymatic digestion during the isolation procedure. Islet viability is affected throughout all steps in this process, from donor death and isolation of islets to culturing and the transplantation process itself. In this thesis a novel strategy to promote islet survival and to re-establish islet vasculature is presented. We show endogenous expression of 51 different genes related to inflammation in cultured islets. Among these genes high expression of MCP-1, MIF, VEGF, thymosin b-10 and IL-8, IL-1β, IL-5R-a, IFN-γ antagonist were found in all donors during the 5- and the 2-day cultures, respectively. Protein expression of these genes can stimulate inflammatory immune responses but also promote tissue repair by attracting curative cells such as endothelial cells (EC) leading to re-establishment of the vasculature. We have established a novel technique by formation of composite islets using EC and mesenchymal stem cells (MSC). EC adhered on the surface of the islets and created a potential blood tolerant surface. The EC-islets showed a degree of protection from the detrimental effects of instant blood-mediated inflammatory reaction (IBMIR) with the major components of IBMIR being decreased in in vitro assays. We combined MSC to the EC-islets with success. The MSC were found to have proliferative effect on EC and the combination of these two cell types on the islets further increased the EC covered surface compared to EC-islets. The EC-MSC-islets in co-culture formed vessel-like structures both into the islets and out to the surrounding matrix. The MSC enhanced the exogenous EC to form vessel-like network in the EC-MSC-islets indicating vascular support by the MSC. The novel strategy and conditions presented herein could alleviate problems related to survival of the islets by promoting revascularization. This would open up a new era in islet transplantation and allow more patients to benefit from this therapy. / Clinical immunology, islet group

The work presented herein focuses on the activation of the adaptive immune system in order to develop T cell-based immunotherapy for viral infections and cancer. The main goal was to identify and activate viral or tumoral antigen-specific T cells by using different identification, isolation and stimulation techniques. One such approach was that we modified dendritic cells (DCs) with an adenoviral vector encoding the full length pp65 antigen from cytomegalovirus (CMV). Through strategic modification techniques we demonstrate that it is possible to obtain DCs presenting antigen-specific peptides both on major histocompatibility complex (MHC) class I and MHC class II molecules for simultaneous CD8+ and CD4+ T cell activation. We also demonstrate that it is possible to generate prostate antigen-specific CD8+ T cells from a naïve repertoire of T cells by using DCs and HLA-A2-restricted peptides derived from prostate tumor-associated antigens or by using an adenoviral vector encoding the full length prostate tumor-associated antigen STEAP. We further demonstrate that CD8+ T cells directed against several prostate-specific peptide epitopes can be found in peripheral blood and in the prostate tumor area of prostate cancer patients. Furthermore, we have characterized a number of prostate-derived cell lines in terms of HLA haplotype and tumor-association antigen expression. We concluded that our methods for generating T cells restricted to CMV antigen have the ability to be applied for adoptive T cell transfer to patients with CMV disease and that prostate antigen-specific T cells can be found within prostate cancer patients, which enables future development of immunotherapeutic strategies for prostate cancer.

T regulatory cells (Tregs) have been extensively studied in patients with cancer or autoimmunity. These cells hamper the immune system’s ability to clear tumor cells in cancer patients. In autoimmune diseases, on the other hand, they are not able to restrain autoreactive immune responses. If we manage to understand Tregs and their role in health and diseases we may be able to develop better immunomodulatory therapies. Early studies demonstrated that tolerance was maintained by a subset of CD25+ T-cells. CD25 was the earliest marker for Tregs and is still often used to define these cells. Several Treg-associated markers have been suggested throughout the years. However, these markers can be upregulated by activated T-cells as well. The most specific marker for Tregs is currently the transcription factor forkhead box P3 (FoxP3). In this thesis, we investigated the presence of CD25- Tregs in patients with B-cell malignancies and in patients with autoimmunity. These cells were identified in both patient groups. Further, patients with B-cell malignancies often have high levels of soluble CD25 (sCD25) in the periphery. In our patient cohorts, the level of peripheral Tregs correlated with the level of sCD25 in patients with lymphoma. Tregs were shown to release sCD25 in vitro and sCD25 had a suppressive effect on T-cell proliferation. These data show that Tregs may release CD25 to hamper T-cell proliferation and that this may be an immune escape mechanism in cancer patients. Previous studies have demonstrated that an increased infiltration of FoxP3+ cells into lymphoma-affected lymph nodes is associated with a better patient outcome. This is in contrast to studies from non-hematological cancers where an increased presence of Tregs is associated with a poor prognosis. Since previous studies have shown that Tregs are able to kill B-cells, we wanted to investigate if Tregs are cytotoxic in patients with B-cell tumors. In the subsequent studies, Tregs from patients with B-cell lymphoma and B-cell chronic lymphocytic leukemia (CLL) were phenotyped to investigate the presence of cytotoxic markers on these cells. FoxP3-expressing T-cells from both patients with CLL and B-cell lymphoma displayed signs of cytotoxicity by upregulation of FasL and the degranulation marker CD107a. Tregs from CLL patients could further kill their autologous B-cells in in vitro cultures. Taken together the studies in this thesis have demonstrated two possible new functions of Tregs in patients with B-cell malignancies and the presence of CD25- Tregs in both cancer and autoimmunity.