Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 8 uppsatser.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [88]-114).

The migration of T lymphocytes is a vital component of the immune system, with roles in immunosurveillance and inflammation. The role of Phosphoinositide 3-kinase within T lymphocyte migration is unclear, with some evidence that it may be a disposable signal. Here, using Staphylococcal Enterotoxin B activated peripheral blood mononuclear cells and the T cell line CEM cells, the role of Phosphoinositide 3-kinase and its downstream kinases was investigated. CCL22 mediated CEM cell migration and CXCL12 mediated peripheral blood mononuclear cell migration were shown to be independent of Phosphoinositide 3-kinase using several different broad-spectrum Phosphoinositide 3-kinase inhibitors. However, these cells were Akt-dependent, as demonstrated by incubation with the Akt inhibitor Akti-1/2. Differences in the effect of the inhibitors on Akt activity were discovered, indicating that either Akt can be activated in the absence of Phosphoinositide 3-kinase, or differences exist regarding the relative abundance of each protein within the cell. Th17 cells are a subtype of the T helper cell family and have been shown to be involved in inflammation and immune diseases. Mouse splenocytes were polarised to a Th17 phenotype and analysed for the surface expression of chemokine receptors. CCR2, CCR6 and CCR9 were shown to be expressed on Th17 cells and upregulated under Th17 polarising conditions. However, only CCR2 and CCR6 induced migration of Th17 cells. This migration was sensitive to Phosphoinositide 3-kinase and Akt inhibitors. This data reveals a model for the migration of Th17 cells to areas of inflammation, and sheds light on the role of Phosphoinositide 3-kinase during this process.

T-lymphocytes (T-cells) trafficking in the lymph nodes (LNs) is key for T-cells activation and their effector functions in adaptive immune responses. T-cells enter the LNs through high endothelial venules (HEVs) and interact with dendritic cells (DCs) for cognate antigens in the T-cell zone (TCZ). After scanning the TCZ for antigens, T-cells leave the LNs through efferent lymphatic vessel. CCR7 and its ligands, CCL19 and CCL21 are involved in the recruitment and compartmentalization of T-cells in LNs. However, their specific role(s) in mediating T-cells migration in LNs sub-regions remain unclear. In addition, the mechanism behind the passage of T-cells from the TCZ to the abluminal side of medullary sinuses (for their exit through medullary sinuses) is not well understood. Here, I hypothesize that different CCL19 and CCL21 fields in LNs sub-regions, orchestrate T-cells sub-regional migration in LNs.. In this study, I examined the CCL19 and CCL21 distribution profiles in mouse LNs sub-regions by immunofluoroscence staining and confocal microscopy. Using microfluidic devices that can flexibly configure well-defined single and co-existing chemical concentration gradients, I quantitatively analyzed the migration of activated human blood T-cells in LNs relevant CCL19 and CCL21 fields. The results suggested a novel CCL19 and CCL21 based combinatorial guiding mechanism for T-cells migration in different LNs sub-regions. In particular, this mechanism operates in the TCZ periphery region to guide T-cells migration away from the TCZ. Furthermore, the CCL19 and CCL21 fields mimicking the region beyond the TCZ toward the medulla result in disturbed chemotaxis, which prevents T-cells from being attracted back to the TCZ. Taken together, this microfluidics-based in vitro study shows the coordinated T-cells migration in different single and combined CCL19 and CCL21 fields, leading to interesting new insights into the guiding mechanisms for T-cells trafficking in LNs sub-regions.

Cardiovascular disease, often induced by hypertension, represents a serious health threat, is a primary cause of death worldwide, and results in altered cardiovascular function and ECM composition. Hypertension and related cardiovascular diseases are associated with immune dysfunction. This dissertation investigated the role of T-lymphocytes in modulating cardiovascular function and ECM composition as a possible therapeutic for the treatment of cardiovascular disease. Study one investigated the role of TCR peptide in the development of hypertension and subsequent cardiovascular changes in Balb/C mice. The coadminstration of TCR and L-NAME/8% NaCl reduced the effects of L-NAME/8% NaCl, decreasing blood pressure and crosslinked collagen compared to L-NAME/8% NaCl alone. Study two examined the effects of T-lymphocyte function on cardiovascular structure and function. Adoptive transfer of T-lymphocytes from C57BL/6 WT mice into C57BL/6 SCID mice induced changes in the SCID so that it resembled the WT donor, with increased percent crosslinked collagen and LOX activity. Hemodynamics in the SCID recipient resembled that of the WT and were significantly different from the sham injected SCID. Study three combined aspects of both previous studies. T-lymphocytes were adoptively transferred from hypertensive WT donors into naïve SCID recipients, who developed hypertension and cardiovascular function resembling the hypertensive donor, as well as changes in the ECM, including increased collagen crosslinking. Study four investigated the effect of strain specific T-lymphocyte polarization on hypertension induced cardiac ECM remodeling. Balb/C, C57BL/6 WT, and C57BL/6 SCID had divergent responses to L-NAME induced hypertension. Ventricular stiffness increased in Balb/C, decreased in C57 SCID and did not change in C57 WT; LOX activity changed correspondingly in all groups. The final study examined the effect of TCR administration on LOX activity and collagen crosslinking. Th1 polarization increased LOX activity and crosslinked collagen with corresponding changes in cardiovascular function. In conclusion, modulation of T-lymphocyte function alters cardiovascular function and ECM composition in pathologic and non-pathologic conditions. Immune modulation should be further investigated as a therapeutic for cardiovascular disease.

Adaptive immune responses are highlyco-ordinated and rely upon efficient intracellular communication to orchestratecell function. Phosphoinositide 3-kinase (PI3K) signalling is a well-studiedand important positive mediator in T lymphocyte function; however the role for SH2-domaincontaining inositol phosphatase 1 (SHIP-1), a negative regulator of PI3Ksignalling, has not been so thoroughly investigated. The use of knockout mousemodels has given an insight into the role of SHIP-1 in murine T cells, butthese are compromised by loss of function during development which impingesupon mature T cell function and by the loss of non-catalytic functions ofSHIP-1. Recent work has indicated a clear rolefor reactive oxygen species (ROS), specifically hydrogen peroxide (H2O2),in immune cell signalling and functional responses including migration. Howeverlike SHIP-1, the functional roles of ROS are poorly understood in human Tlymphocytes, particularly the mechanisms by which ROS signals to alter Tlymphocyte biology. ROS has been previously shown to activate PI3K, Mitogenactivate protein kinase (MAPK) and Src family tyrosine kinase (SFK) signallingin a number of different cell types. In addition, ROS have been shown to inactivatephosphatase and tensin homology (PTEN), another negative regulator of PI3K, andare postulated to inactive SHIP-1 signalling. A pharmacological approach wasutilised to manipulate the catalytic activity of SHIP-1 and the cellularaccumulation of ROS in primary human T lymphocytes. Remarkably, it wasdetermined that both SHIP-1 activation and inhibition reduced the ligand-mediatedfunctions of human T lymphocytes, including signalling, proliferation, adhesionand migration. Furthermore, H2O2 selectively inhibited directionalmigration to chemokine CXCL11, enhanced F-actin polymerisation and decreasedactin polarisation to CXCL11. H2O2 required SFKsignalling to induce the phosphorylation/catalytic activation of SHIP-1 and todecrease the surface expression of CXCR3, both of which could be mechanismsunderlying the deficiency in migration observed with H2O2.

Abnormal T lymphocyte reactions in both autism and Alzheimer's disease (AD) have been reported. This research investigated the possibility that these abnormalities may involve circulating antithymic antibodies. Plasma samples from autistic patients, AD patients, and normal-matched controls were tested for reactivity against murine thymocytes. In the first of 3 studies results of the enzyme-linked immunosorbent assay (ELISA) were statistically significant for binding (P < 0.001) between antithymic antibodies in plasmas of AD patients and murine thymocytes. Binding (P < 0.05) in low dilutions (1/2.5 and 1/5} of autistic patient plasmas was also observed. In the second study, plasmas of neither autistic nor AD patients significantly inhibited DNA synthesis of thymic cells in the presence of interleukin-1 (IL-l} and phytohemagglutinin (PHA). In the third study, no significant increases (P > 0.05) in cytotoxic activities were detected using AD patient plasmas and both untreated and heat-treated autistic patient plasmas. After further testing, these heat-treated plasmas diluted 1/64 and 1/128 had increased cytotoxicities (P Therefore, circulating antithymic antibodies may be involved in abnormal T lymphocyte reactions in autism and AD. Since they probably do not act alone, future research should study these complex abnormalities using human thymocytes.

Although melanoma accounts for only 4% of skin cancers, it is responsible for 80% of deaths from skin cancers and its incidence in Caucasians has been increasing steadily during the last 30 years. So far, no treatment - except surgery at the earliest stages of the disease - has been shown to significantly improve survival. New treatments are thus clearly needed. The interest of immunologists for melanoma is based on particular features of this tumor. Rare spontaneous regressions have been described, which are possibly mediated by immune responses. Moreover, melanoma cell lines are relatively easy to obtain, providing essential tools for laboratory studies. The first melanoma vaccines involved inoculations of patients with autologous or allogeneic melanoma cells, as well as a variety of immunological adjuvants. Since the beginning of the nineties, the identification of antigens recognized on human tumors by autologous T lymphocytes has opened the way for new vaccination strategies involving molecularly defined tumor antigens. An important group of antigens recognized by T lymphocytes is encoded by “cancer-germ line genes”, which are expressed in tumors of various histological types, but are silent in normal tissues, with the exception of testis germinal cells and placental trophoblast. Since the latter do not express the HLA molecules required to present these antigens to the T lymphocytes, cancer-germ line genes encoded antigens are only present on tumors, which should limit the risk of generating autoimmune diseases as a consequence of vaccination. Therefore, these widely shared tumor specific antigens should represent good targets for the development of cancer vaccines. Our clinical research program of therapeutic vaccinations focuses on antigens encoded by the MAGE family of cancer-germ line genes. Most of the patients included in our phase I/II immunization trials had measurable metastatic melanoma. Several MAGE peptides as well as a recombinant MAGE-3 protein have been tested, while several additional trials are ongoing, including immunizations with a recombinant poxvirus coding for 2 MAGE epitopes. No major toxicity was reported. Tumor regressions have been observed in a minority of patients, mainly those who had regional or distant metastases without visceral involvement. Some of these regressions have been complete and long lasting. Although the rate of objective tumor response observed is low, it is clearly higher than the rate of spontaneous tumor regression observed in melanoma. Other immunization modes against T-cell defined epitopes are currently being explored by several groups in human clinical trials. Vaccines include peptides presented by class I or class II HLA molecules, proteins given alone or mixed with immunological adjuvants or cytokines, recombinant viral or bacterial vectors, dendritic cells and DNA encoding the antigen. Adoptive transfer of T lymphocytes selected for their capacity to recognize defined epitopes presented by the tumor represents another type of approach aiming at the destruction of the tumor by the immune system. It is difficult to predict whether and when therapeutic vaccination against cancer will reach an efficacy that will be sufficient for a standard cancer treatment. Provided their low toxicity, these vaccines should be tested in an adjuvant setting, at earlier stages of the disease.

Recent studies support the association between metabolic syndrome (MetS), a cluster of cardiovascular risk factors, and diastolic dysfunction. Disproportionate collagen accumulation, particularly cross-linking of collagen, plays a key role in translating interstitial fibrosis into mechanical chamber stiffness and diastolic dysfunction. Characteristic changes in the expression and activity of myocardial lysyl oxidase (LOX), a matrix modifying enzyme that catalyzes cross-linked collagen, are unclear in MetS. We established a diet-induced MetS model to study diastolic dysfunction by treating male C57BL/6 mice a high-fat high-simple carbohydrate (HFHSC) diet for 6 months. Despite blunted gene expression of LOX isoforms, MetS mice demonstrated significant increase in the ratio of protein expression of mature to proenzyme LOX, enhanced LOX activity, and increased cardiac cross-linked collagen compared with controls. This fibrotic response coincided with marked increase in left ventricular end-diastolic pressure and stiffness and impaired diastolic filling pattern. Our data demonstrate that diet-induced MetS alters the remodeling enzyme LOX, thereby increasing the amount of crosslinking and inducing diastolic dysfunction.Furthermore we examined the role of T-lymphocytes in myocardial LOX regulation in diet-induced fibrotic hearts. Female SCID mice which are devoid of functional T-lymphocytes and C57BL/6 mice were treated with HFHSC diet for 12 months. Similar to male C67BL/6, female HFHSC-fed C57BL/6 mice demonstrated significant increase in maturation and catalytic activity of myocardial LOX, cross-linking, ventricular stiffness and diastolic dysfunction. Whereas induction of LOX protein was minimal in SCID mice compared with wild-type counterparts. Correspondingly fibrillar cross-linked collagen formation and diastolic dysfunction were less prominent in SCID mice. Our results suggest a potential role of T-lymphocytes in induction of myocardial stiffness and diastolic dysfunction through modulation of LOX-dependent collagen maturation.Moreover we studied the role of leptin, an adipokine over-produced in MetS with fibrotic effects in non-cardiac tissues, as a key mediator of profibrogenic responses in the heart by administrating leptin to C57BL/6 and leptin-deficient ob/ob mice. With exogenous leptin administration ob/ob mice displayed passive diastolic filling dysfunction that coincided with increase in myocardial collagen compared with ob/ob controls. Our findings suggest profibrotic effects of leptin in the heart, primarily through predominance of collagen synthesis over degradation.

The proteoglycan CD44 is a widely expressed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, and is involved in processes ranging from metastasis to wound healing. In the immune system, leukocyte activation induces hyaluronan binding through changes in CD44 post-translational modification, but these changes have not been well characterized. Here I identify chondroitin sulfate addition to CD44 as a negative regulator of hyaluronan binding. Chondroitin sulfate addition was analyzed by sulfate incorporation and Western blotting and determined to occur at serine 180 in human CD44 using site-directed mutagenesis. Mutation of serine 180 increased hyaluronan binding by both a CD44-immunoglobulin fusion protein expressed in HEK293 cells, and full-length CD44 expressed in murine L fibroblast cells. In bone marrow-derived macrophages, hyaluronan binding induced by the inflammatory cytokines tumor necrosis factor-α and interferon-γ corresponded with reduced chondroitin sulfate addition to CD44. Retroviral infection of CD44⁻/⁻ macrophages with mouse CD44 containing a mutation at serine 183, equivalent to serine 180 in human CD44, resulted in hyaluronan binding that was constitutively high and no longer enhanced by stimulation. These results demonstrate that hyaluronan binding by CD44 is regulated by chondroitin sulfate addition in macrophages. A functional consequence of altered chondroitin sulfate addition and increased hyaluronan binding was observed in Jurkat T cells, which became more susceptible to activation-induced cell death when transfected with mutant CD44. The extent of cell death was dependent upon both the hyaluronan binding ability of CD44 and the size of hyaluronan itself, with high molecular mass hyaluronan having a greater effect than intermediate or low molecular mass hyaluronan. The addition of hyaluronan to pre-activated Jurkat T cells induced rapid cell death independently of Fas and caspase activation, identifying a unique Fas-independent mechanism for inducing cell death in activated cells. Results were comparable in splenic T cells, where high hyaluronan binding correlated with increased phosphatidylserine exposure, and hyaluronan-dependent cell death occurred in a population of restimulated cells in the absence of Fas-dependent cell death. Together these results reveal a novel mechanism for regulating hyaluronan binding and demonstrate that altered chondroitin sulfate addition can affect CD44 function.