A hallmark of chronic human immunodeficiency virus (HIV) infection is the impairment of CD8 T cell survival and effector functions, which likely contributes to HIV pathogenesis. A number of factors could be attributed to this impairment, including the declining number of CD4 T cells, progressive destruction of secondary lymphoid tissues and an increasingly inhibitory environment. As highly active antiretroviral therapy shows limited efficacy in improving CD8 T cell functions, this thesis explores the therapeutic application of costimulatory molecules in directly stimulating non-functional HIV-specific CD8 T cells and ultimately their relevance to the control of chronic HIV infection. Costimulatory molecules are adjuvants for functional activation of T cells that act in concert with the antigen-specific signal. The Tumor Necrosis Factor (TNF) family member, 4-1BBL, emerges as the most effective costimulatory molecule in the antigen-specific expansion of human memory CD8 T cells as compared to the related TNF family members CD70 and LIGHT. As well, 4-1BBL improves the cytolytic function of T lymphocytes on a per cell basis. Furthermore, 4-1BBL is identified as a key component in the therapeutic rescue of CD8 T cell function and its effect is at least partially dependent on its signaling adaptor TNF receptor associated factor 1 (TRAF1), both in vitro and in vivo. This thesis also identifies the loss of TRAF1 as a new mechanism of immune dysregulation of HIV-specific CD8 T cells during the chronic phase of HIV infection and offers a means to correct it. The loss of TRAF1 has functional relevance in HIV suppression and HIV-specific CD8 T cell responses. Finally, a combination therapy involving agonistic anti-4-1BB antibody is shown to be successful in a proof of concept treatment of chronic lymphocytic chroriomeningitis virus (LCMV) infection in mice, resulting in sustained reduction in viral load. A new model of HIV-specific CD8 T cell dysfunction is constructed based on these findings.

A hallmark of chronic human immunodeficiency virus (HIV) infection is the impairment of CD8 T cell survival and effector functions, which likely contributes to HIV pathogenesis. A number of factors could be attributed to this impairment, including the declining number of CD4 T cells, progressive destruction of secondary lymphoid tissues and an increasingly inhibitory environment. As highly active antiretroviral therapy shows limited efficacy in improving CD8 T cell functions, this thesis explores the therapeutic application of costimulatory molecules in directly stimulating non-functional HIV-specific CD8 T cells and ultimately their relevance to the control of chronic HIV infection. Costimulatory molecules are adjuvants for functional activation of T cells that act in concert with the antigen-specific signal. The Tumor Necrosis Factor (TNF) family member, 4-1BBL, emerges as the most effective costimulatory molecule in the antigen-specific expansion of human memory CD8 T cells as compared to the related TNF family members CD70 and LIGHT. As well, 4-1BBL improves the cytolytic function of T lymphocytes on a per cell basis. Furthermore, 4-1BBL is identified as a key component in the therapeutic rescue of CD8 T cell function and its effect is at least partially dependent on its signaling adaptor TNF receptor associated factor 1 (TRAF1), both in vitro and in vivo. This thesis also identifies the loss of TRAF1 as a new mechanism of immune dysregulation of HIV-specific CD8 T cells during the chronic phase of HIV infection and offers a means to correct it. The loss of TRAF1 has functional relevance in HIV suppression and HIV-specific CD8 T cell responses. Finally, a combination therapy involving agonistic anti-4-1BB antibody is shown to be successful in a proof of concept treatment of chronic lymphocytic chroriomeningitis virus (LCMV) infection in mice, resulting in sustained reduction in viral load. A new model of HIV-specific CD8 T cell dysfunction is constructed based on these findings.

Monoclonal B lymphocytosis (MBL) has been recognized as a novel diagnostic condition. This study aims at the identification of clonal lymphocytosis in the patients with asymptomatic lymphocytosis. A total of 203 patients were evaluated for clonal B and T lymphocytosis by using flow cytometry and multiplex-PCR. Among them clonal B- or T-cells were detected in 54.2% of the cases, of which 38.4% were clonal B-cells and 15.8% were clonal T-cells cases. By immunophenotype, MBL was classified into the chronic lymphocytic leukemia (CLL) type (21.7%) and non-CLL-type (7.4%). Flow cytometry analysis and cell counts were used to determine the size of clonal population, and the data indicate that MBL and CLL are present in a continuous spectrum of clonal expansion. The findings may contribute to the current understanding of MBL and evaluation of incidental lymphocytosis. Further studies are required to evaluate clonal progression as a precursor stage of lymphoid malignancy.

Natürlich vorkommende regulatorische T-Zellen spielen eine entscheidende Rolle für die intestinale Immunhomöostase und Limitierung von (Auto)-Immunität. Sie exprimieren den Transkriptionsfaktor Foxp3 und an der Oberfläche die α-Kette des IL-2 Rezeptors (CD25). Im Tiermodell verhindern regulatorische T-Zellen Autoimmunopathien, Transplantatabstoßungen und entzündliche Darmerkrankungen. Da Foxp3+ regulatorische T-Zellen nur äußerst geringe Mengen an Interleukin-2 synthetisieren, sind sie auf eine adäquate Versorgung angewiesen. Konventionelle T-Zellen werden als bedeutende IL-2 Quelle für Treg-Zellen vermutet, doch über die Mechanismen und räumlich-zeitliche Dynamik der Treg-Effektor-Zellinteraktion ist bisher nur wenig bekannt. In dieser Arbeit wurden Foxp3+ regulatorische T-Zellen in Mausgeweben analysiert und Zellinteraktionen mit Effektorzellen im Darm charakterisiert. Es wurde ein theoretisches Modell zur Evaluierung von Zell-Zellkontakten erarbeitet und experimentell überprüft. Es konnte gezeigt werden, dass in der Akutphase einer T-Zell-induzierten Kolitis und im Kolon von gesunden Wildtyp-Mäusen Foxp3+ regulatorische T-Zellen an Ki-67+ proliferierenden T-Zellen akkumulieren. Diese Zellinteraktionen sind abhängig von Interleukin-2, da IL-2 defiziente Mäuse keine signifikanten Treg-Effektor-Zellakkumulationen aufweisen. Die Analyse der Genexpression konnte zeigen, dass Ki-67+ Zellen Interleukin-2 produzieren. Lokal sezerniertes Interleukin-2 könnte als Sensor für Entzündungsprozesse chemotaktisch auf Foxp3+ regulatorische T-Zellen wirken und die Akkumulation an proliferierenden, IL-2 produzierenden Effektorzellen bedingen. Dieser Mechanismus könnte einerseits zur lokalen Versorgung mit IL-2 dienen und gleichzeitig regulierend auf Effektorzellen in unmittelbarer Umgebung wirken. Dieser Prozess würde zur Erhaltung von regulatorischen T-Zellen in der Peripherie und zur Sicherung der intestinalen Immunbalance beitragen.

The connection between T cell activation, plasma membrane order and actin filament dynamics was the main focus of this study. Laurdan and di-4-ANEPPDHQ, membrane order sensing probes, were shown to report only on lipid packing rather than being influenced by the presence of membrane-inserted peptides justifying their use in membrane order studies. These dyes were used to follow plasma membrane order in live cells at 37°C. Disrupting actin filaments had a disordering effect while stabilizing actin filaments had an ordering effect on the plasma membrane, indicating there is a basal level of ordered domains in resting cells. Lowering PI(4,5)P2 levels decreased the proportion of ordered domains strongly suggesting that the connection of actin filaments to the plasma membrane is responsible for the maintaining the level of ordered membrane domains. Membrane blebs, which are detached from the underlying actin filaments, contained a low fraction of ordered domains. Aggregation of membrane components resulted in a higher proportion of ordered plasma membrane domains and an increase in cell peripheral actin polymerization. This strongly suggests that the attachment of actin filaments to the plasma membrane induces the formation of ordered domains. Limited cholesterol depletion with methyl-beta-cyclodextrin triggered peripheral actin polymerization. Cholesterol depleted cells showed an increase in plasma membrane order as a result of actin filament accumulation underneath the membrane. Moderate cholesterol depletion also induced membrane domain aggregation and activation of T cell signaling events. The T cell receptor (TCR) aggregation caused redistribution of domains resulting in TCR patches of higher order and the bulk membrane correspondingly depleted of ordered domains. This suggests the preexistence of small ordered membrane domains in resting T cells that aggregate upon cell activation. Increased actin polymerization at the TCR aggregation sites showed that actin polymerization is strongly correlated with the changes in the distribution of ordered domains. The distribution of the TCR in resting cells and its colocalization with actin filaments is cell cycle dependent. We conclude that actin filament attachment to the plasma membrane, which is regulated via PI(4,5)P2, plays a crucial role in the formation of ordered domains. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript.

This thesis investigated the number and function of natural killer T-cells (NK T-cells) as a function of age, in coeliac disease, Crohn’s disease and ulcerative colitis. NK T-cells are a newly appreciated class of immune cells that are able to regulate the activity of the broader T-cell population. NK T-cells have been implicated in animal models of autoimmune disease and in human autoimmune disease. A subset of NK cells express the T-cell receptor (TCR) and are termed NK T-cells. In humans a further small subset of NK T-cells express an invariant TCR α chain (Vα24Jα18) and contain the immunoregulatory cell population that is distinguished from classical T-cells by promptly producing interleukin-4 (IL-4). Invariant NK T-cells (iNK T-cells) have the surface phenotype of Vα24+ Vβ11+ T-cells and express CD161+ NK markers. They are CD4+ (single positive; SP) or CD4- (double negative; DN), CD1d restricted and are α-galactosylceramide (α-GalCer) reactive. NKT cells have been implicated in numerous autoimmune disorders. Early work showed a major deficiency of NKT cell numbers in nonobese diabetic (NOD) mice, a well-established model of spontaneous, autoimmune T-cell mediated insulin-dependent diabetes. Both the number of NKT cells and function, as assessed by IL-4 release following TCR ligation, are dramatically reduced in NOD mice. NK T-cells have been implicated in other models of autoimmunity such as, experimental allergic encephalomyelitis (EAE). They have since been investigated and shown to be deficient in a number of human autoimmune diseases including, systemic sclerosis (SSc), and systemic lupus erythematosus (SLE), multiple sclerosis, atopic asthma, atopic dermatitis, rheumatoid arthritis, type 1 diabetes mellitus and scleroderma. The basis of the work presented within this thesis originated from the deficiency of NK T-cells in models of autoimmune diseases and human autoimmune diseases. The initial aim of this thesis was to investigate the phenotype and function of Vα24+ NK T-cells in normal healthy control subjects and with respect to age. The original aim was to investigate whether NK cells, T-cells, NK T-like cells and invariant NK T-cells (iNK T-cells) are deficient in coeliac disease, Crohn’s disease and/or ulcerative colitis. Blood was collected for flow cytometry from normal control subjects, subjects with coeliac disease, Crohn’s disease and ulcerative colitis. The number of circulating NK cells, T-cells, NK T-like cells and iNK T-cells was assessed by three-colour flow cytometry. Intracellular cytokine production was measured after in vitro anti-CD3/ anti-CD28 antibodies, gluten fraction 3 and PMA:ionomycin stimulation. Vα24+ T-cells were quantified in ileocolonic biopsies by immunofluorescence and as mRNA by relative and real-time PCR (RT-PCR). The number of circulating Vα24+ T-cells and iNK T-cells decrease with age in normal healthy control subjects. Cytokine production was also affected by age. The work of this thesis has identified a subpopulation of otherwise normal healthy individuals whom have normal numbers of circulating Vα24+ T-cells, reduced numbers of circulating Vα24+ Vβ11+ T-cells and consequently iNK Tcells. Circulating CD161+ NK cells, Vα24+ T-cells and the SP subset of Vα24+ Tcells were reduced in coeliac disease. The low numbers of circulating Vα24+ T-cells was independent of diet. The number of circulating Vα24+ Vβ11+ Tcells were reduced in coeliac disease, and as a consequence, the number of circulating Vα24+ Vβ11+ α-GalCer/CD1d tetramer+ and Vα24+ 6B11+ iNK T-cells were reduced. The deficiency of Vα24+ T-cells was not confined to the blood, but observed within the intestinal mucosa. Intestinal Vα24 mRNA expression from subjects with coeliac disease was reduced compared to levels in normal subjects as assessed by relative and RT-PCR. Thus, Vα24+ T-cells were deficient in coeliac disease both systemically and mucosally. Cytokine production by Vα24+ T-cells, 6B11+ and Vα24+ α-GalCer/CD1d tetramer+ iNK T-cells after 4 h in vitro anti-CD3 stimulation was also impaired in subjects with coeliac disease. Circulating CD56+, CD57+, CD94+, CD161+ NK cells were reduced in Crohn’s disease and ulcerative colitis. Vα24+ T-cells and the SP subset of Vα24+ T-cells were reduced in Crohn’s disease but not in ulcerative colitis. Circulating Vα24+ Vβ11+ T-cells, Vα24+ Vβ11+ α-GalCer/CD1d tetramer+ and Vα24+ 6B11+ iNK T-cells were deficient in both Cohn’s disease and ulcerative colitis. The deficiency of Vα24+ T-cells was also observed within the intestinal mucosa. Intestinal Vα24 mRNA expression from Crohn’s disease and ulcerative colitis was reduced compared to levels in normal subjects as assessed by relative and RT-PCR. Cytokine production by Vα24+ T-cells, 6B11+ and Vα24+ α-GalCer/CD1d tetramer+ iNK T-cells after 4 h in vitro anti-CD3 stimulation was impaired for subjects with Crohn’s disease and ulcerative colitis. In summary, Vα24+ T-cell number and function were affected by age. Further investigations are warranted to see if deficiency of this immunoregulatory population is associated with disease. The decrease and dysfunction in immunoregulatory cells, Vα24 T-cells and iNK T-cells could contribute to the pathogenesis of coeliac disease, Crohn’s disease and ulcerative colitis. Coeliac disease, Crohn’s disease and ulcerative colitis are polygenetic diseases in which environmental factors play a significant role in disease development and state. The reduced numbers of iNK T-cell along with their impaired function may only be two factors. Presumably, other factors are involved. Nevertheless, iNK T-cells offer a potential target for the therapeutic intervention of coeliac disease, ulcerative colitis and Crohn’s disease. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1345088 / Thesis (Ph.D.) - University of Adelaide, School of Medicine, 2008

This thesis investigated the number and function of natural killer T-cells (NK T-cells) as a function of age, in coeliac disease, Crohn’s disease and ulcerative colitis. NK T-cells are a newly appreciated class of immune cells that are able to regulate the activity of the broader T-cell population. NK T-cells have been implicated in animal models of autoimmune disease and in human autoimmune disease. A subset of NK cells express the T-cell receptor (TCR) and are termed NK T-cells. In humans a further small subset of NK T-cells express an invariant TCR α chain (Vα24Jα18) and contain the immunoregulatory cell population that is distinguished from classical T-cells by promptly producing interleukin-4 (IL-4). Invariant NK T-cells (iNK T-cells) have the surface phenotype of Vα24+ Vβ11+ T-cells and express CD161+ NK markers. They are CD4+ (single positive; SP) or CD4- (double negative; DN), CD1d restricted and are α-galactosylceramide (α-GalCer) reactive. NKT cells have been implicated in numerous autoimmune disorders. Early work showed a major deficiency of NKT cell numbers in nonobese diabetic (NOD) mice, a well-established model of spontaneous, autoimmune T-cell mediated insulin-dependent diabetes. Both the number of NKT cells and function, as assessed by IL-4 release following TCR ligation, are dramatically reduced in NOD mice. NK T-cells have been implicated in other models of autoimmunity such as, experimental allergic encephalomyelitis (EAE). They have since been investigated and shown to be deficient in a number of human autoimmune diseases including, systemic sclerosis (SSc), and systemic lupus erythematosus (SLE), multiple sclerosis, atopic asthma, atopic dermatitis, rheumatoid arthritis, type 1 diabetes mellitus and scleroderma. The basis of the work presented within this thesis originated from the deficiency of NK T-cells in models of autoimmune diseases and human autoimmune diseases. The initial aim of this thesis was to investigate the phenotype and function of Vα24+ NK T-cells in normal healthy control subjects and with respect to age. The original aim was to investigate whether NK cells, T-cells, NK T-like cells and invariant NK T-cells (iNK T-cells) are deficient in coeliac disease, Crohn’s disease and/or ulcerative colitis. Blood was collected for flow cytometry from normal control subjects, subjects with coeliac disease, Crohn’s disease and ulcerative colitis. The number of circulating NK cells, T-cells, NK T-like cells and iNK T-cells was assessed by three-colour flow cytometry. Intracellular cytokine production was measured after in vitro anti-CD3/ anti-CD28 antibodies, gluten fraction 3 and PMA:ionomycin stimulation. Vα24+ T-cells were quantified in ileocolonic biopsies by immunofluorescence and as mRNA by relative and real-time PCR (RT-PCR). The number of circulating Vα24+ T-cells and iNK T-cells decrease with age in normal healthy control subjects. Cytokine production was also affected by age. The work of this thesis has identified a subpopulation of otherwise normal healthy individuals whom have normal numbers of circulating Vα24+ T-cells, reduced numbers of circulating Vα24+ Vβ11+ T-cells and consequently iNK Tcells. Circulating CD161+ NK cells, Vα24+ T-cells and the SP subset of Vα24+ Tcells were reduced in coeliac disease. The low numbers of circulating Vα24+ T-cells was independent of diet. The number of circulating Vα24+ Vβ11+ Tcells were reduced in coeliac disease, and as a consequence, the number of circulating Vα24+ Vβ11+ α-GalCer/CD1d tetramer+ and Vα24+ 6B11+ iNK T-cells were reduced. The deficiency of Vα24+ T-cells was not confined to the blood, but observed within the intestinal mucosa. Intestinal Vα24 mRNA expression from subjects with coeliac disease was reduced compared to levels in normal subjects as assessed by relative and RT-PCR. Thus, Vα24+ T-cells were deficient in coeliac disease both systemically and mucosally. Cytokine production by Vα24+ T-cells, 6B11+ and Vα24+ α-GalCer/CD1d tetramer+ iNK T-cells after 4 h in vitro anti-CD3 stimulation was also impaired in subjects with coeliac disease. Circulating CD56+, CD57+, CD94+, CD161+ NK cells were reduced in Crohn’s disease and ulcerative colitis. Vα24+ T-cells and the SP subset of Vα24+ T-cells were reduced in Crohn’s disease but not in ulcerative colitis. Circulating Vα24+ Vβ11+ T-cells, Vα24+ Vβ11+ α-GalCer/CD1d tetramer+ and Vα24+ 6B11+ iNK T-cells were deficient in both Cohn’s disease and ulcerative colitis. The deficiency of Vα24+ T-cells was also observed within the intestinal mucosa. Intestinal Vα24 mRNA expression from Crohn’s disease and ulcerative colitis was reduced compared to levels in normal subjects as assessed by relative and RT-PCR. Cytokine production by Vα24+ T-cells, 6B11+ and Vα24+ α-GalCer/CD1d tetramer+ iNK T-cells after 4 h in vitro anti-CD3 stimulation was impaired for subjects with Crohn’s disease and ulcerative colitis. In summary, Vα24+ T-cell number and function were affected by age. Further investigations are warranted to see if deficiency of this immunoregulatory population is associated with disease. The decrease and dysfunction in immunoregulatory cells, Vα24 T-cells and iNK T-cells could contribute to the pathogenesis of coeliac disease, Crohn’s disease and ulcerative colitis. Coeliac disease, Crohn’s disease and ulcerative colitis are polygenetic diseases in which environmental factors play a significant role in disease development and state. The reduced numbers of iNK T-cell along with their impaired function may only be two factors. Presumably, other factors are involved. Nevertheless, iNK T-cells offer a potential target for the therapeutic intervention of coeliac disease, ulcerative colitis and Crohn’s disease. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1345088 / Thesis (Ph.D.) - University of Adelaide, School of Medicine, 2008

The Mater Medical Research Institute proposes to undertake a Phase I clinical trial using CMRF-56 immunoselected blood dendritic cells (BDC) loaded with control and myeloma-associated tumour peptide antigens for the treatment of multiple myeloma (MM) patients with minimal residual disease. This thesis describes some of the fundamental pre-clinical in vitro experiments undertaken in preparation for this trial so as to maximise the potential of this vaccine to induce myeloma-specific immune responses. These experiments involved determining the parameters for optimal activation of the CMRF-56 immunoselected cell preparation and exploring the potential of novel myeloma peptide antigens to induce anti-myeloma cytotoxic T lymphocyte (CTL) responses. CMRF-56 immunoselected cell preparations, containing predominately myeloid BDC, monocytes and B cells, were prepared from both healthy donors and myeloma patients. Activation of this preparation with granulocyte macrophage colony stimulating factor (GM-CSF) was found to increase co-stimulatory molecule expression by and survival of BDC, improve peptide- and lysate-specific CTL induction, and, in combination with prostaglandin E2 (PGE2), improve chemokine-specific migration of BDC. Following optimisation of in vitro CTL generation protocols, GM-CSF activated CMRF-56 immunoselected cells were examined for their ability to induce myeloma-specific immunity. Using lysate from myeloma cell line U266 as an antigen source, a polyclonal T cell pool was generated within which peptide specific CTL recognising myeloma antigens Muc1, HM1.24/BST2, DKK-1 and CT-7/MAGE-C1 could be identified. Furthermore, GM-CSF activated CMRF-56 immunoselected cells pulsed with HLA-A*201 restricted peptides derived from Muc1, HM1.24/BST2 and CT-7/MAGE-C1 could induce CTL capable of lysing both peptide- and myeloma cell line targets in both healthy donors and myeloma patients. These results provide the first evidence of immunogenic HLA-A*201 restricted epitopes of novel myeloma antigen CT-7/MAGE-C1. The data collected in this study supports the application of GM-CSF activated CMRF-56 immunoselected cells loaded with defined myeloma peptide antigens for the therapeutic vaccination of MM patients with minimal residual disease.

The Mater Medical Research Institute proposes to undertake a Phase I clinical trial using CMRF-56 immunoselected blood dendritic cells (BDC) loaded with control and myeloma-associated tumour peptide antigens for the treatment of multiple myeloma (MM) patients with minimal residual disease. This thesis describes some of the fundamental pre-clinical in vitro experiments undertaken in preparation for this trial so as to maximise the potential of this vaccine to induce myeloma-specific immune responses. These experiments involved determining the parameters for optimal activation of the CMRF-56 immunoselected cell preparation and exploring the potential of novel myeloma peptide antigens to induce anti-myeloma cytotoxic T lymphocyte (CTL) responses. CMRF-56 immunoselected cell preparations, containing predominately myeloid BDC, monocytes and B cells, were prepared from both healthy donors and myeloma patients. Activation of this preparation with granulocyte macrophage colony stimulating factor (GM-CSF) was found to increase co-stimulatory molecule expression by and survival of BDC, improve peptide- and lysate-specific CTL induction, and, in combination with prostaglandin E2 (PGE2), improve chemokine-specific migration of BDC. Following optimisation of in vitro CTL generation protocols, GM-CSF activated CMRF-56 immunoselected cells were examined for their ability to induce myeloma-specific immunity. Using lysate from myeloma cell line U266 as an antigen source, a polyclonal T cell pool was generated within which peptide specific CTL recognising myeloma antigens Muc1, HM1.24/BST2, DKK-1 and CT-7/MAGE-C1 could be identified. Furthermore, GM-CSF activated CMRF-56 immunoselected cells pulsed with HLA-A*201 restricted peptides derived from Muc1, HM1.24/BST2 and CT-7/MAGE-C1 could induce CTL capable of lysing both peptide- and myeloma cell line targets in both healthy donors and myeloma patients. These results provide the first evidence of immunogenic HLA-A*201 restricted epitopes of novel myeloma antigen CT-7/MAGE-C1. The data collected in this study supports the application of GM-CSF activated CMRF-56 immunoselected cells loaded with defined myeloma peptide antigens for the therapeutic vaccination of MM patients with minimal residual disease.

Abstract Early in the evolution of the vertebrates it is thought that two genomic duplications occurred, providing a basis for the evolution in body plan and neural crest of very early vertebrates and substantive material for further evolution of various gene families such as those making up a number of components of the adaptive vertebrate immune system. While the bony fish possibly had another, genome duplications are not generally a feature of vertebrate evolution and indeed the appearance of an antigen-adaptive immune recognition system may have served to limit the size that various vertebrate genomes, including that of the human, can in fact achieve. This initial step in vertebrate immune evolution, the establishment of recognition of non-self against the unique set of 'self' epitopes for an individual, provided an immensely powerful weapon in immune function with the ability to tailor a defense against as-yet-unseen dangers at any time albeit with the pitfall of autoimmune disease. As the recognition sites of the antigen receptor molecules such as TcR are produced by clonal modification of the segments provided in the germline and are thus not in the genome itself, pathogens have not been able to hijack this one component of the immune system in the way so many other components have been put to use throughout evolution, nor do these components necessarily reveal themselves as associated with disease through genome screens. Importantly, overall immune function is determined not just by the potential repertoire of recognition receptors but also by the ability of immunocompetent cells to migrate in a tissue specific fashion through the use of various chemokines and their receptors. Typical of the hijacking of an immune system component by a pathogen is the use of a chemokine ligand gene in the viral ancestor to SIV and HIV, allowing for virus binding to immunocompetent cells as is seen in the use of the CCR5 chemokine receptor by macrophage-tropic HIV strains. This thesis describes the allele and genotype frequencies for several TcR beta-chain variable segment polymorphisms in a population of MS patients compared with controls before and after stratification for HLA-DR15, polymorphism in the Apo-1 / Fas promoter, the DRB1 Val86/Val86 genotype, CCR5-delta32 and the HLA-DRA promoter. The thesis continues with CCR5-delta32 genotyping in IDDM, MS and SLE cohorts and then examines the question of the population of origin of the delta-32 allele of the CCR5 receptor for chemokine. Here, a case / control comparison of 122 RR-MS patients with 96 normal individuals was made for allele and genotype frequencies and for haplotypes formed by pairs of TCRB markers. Further analysis was made after HLA-DR15 stratification. Linkage disequilibrium was found between pairs of alleles of bv8s1, bv10s1, bv15s1 and bv3s1 loci in both patients and controls. In the RR-MS cohort, an increase in the allele frequency of bv8s1*2 was seen (p = 0.03) and the haplotype bv8s1*2 / bv3s1*1 was increased (p = 0.006), and both were found to be statistically significant. In the DR15-positive group, association between MS and TCRB was seen with the bv8s1*2 allele (p = 0.05) and the bv8s1*2 / bv10s1 haplotypes (p = 0.048), while the haplotype associations seen among the DR15-negative patients included the bv3s1*1 allele (bv10s1*1 / bv3s1*1, p = 0.022; bv8s1*2 / bv3s1*1, p = 0.048). While no associations were found after stratification for SDF1-3'A, Apo-1 / Fas or DRB1 there were modest interactions between bv3s1, bv10s1 and bv15s1 and the HLA-DRA promoter. These results support the involvement of the TCRB region in MS susceptibility. The further study of autoimmune disease here includes genotype analysis of CCR5-delta32 in type 1 diabetes (IDDM) and SLE. CCR5 is the major co-receptor for viral entry used by macrophage-tropic HIV strains and protection from infection is seen in homozygotes for CCR5-delta32. In diabetes, infiltration of pancreatic tissue by autoreactive T-cells involves secretion of multiple cytokines and chemokine receptor expression. Variation in the chemokine receptor CCR5 may result in differences in inflammatory cell migration in response to relevant chemokines. Adolescents with type 1 diabetes were genotyped for CCR5-delta32 (n = 626). The allele frequency was compared with that of 253 non-diabetic adolescents and with that of 92 adults with SLE. A reduced allele frequency was seen in type 1 diabetes compared with controls (0.092 vs 0.123, p = 0.05). This difference was not seen for the cohort of patients with SLE (freq = 0.114). A reduction in the number of CCR5-delta32/delta32 homozygotes, who lack CCR5, in the type 1 diabetes cohort was also seen and while not statistically significant (2 observed compared to 5.25 expected; p = 0.12) is interesting. These results suggest a partial protection from type 1 diabetes for CCR5-delta32 homozygous individuals is possible and that CCR5 has a potential role in the pathogenesis of type 1 diabetes. Global surveys of the CCR5-delta32 allele have confirmed a single mutation event in a Northeastern European population as the source of this allele. Here, Australian Ashkenazi Jews (n = 807) were found to have a CCR5-delta32 allele frequency of 14.6% while Australian Sephardic Jews (n = 35) had a frequency of 5.7% and non-Jewish Australian controls (n = 311) had an allele frequency of 11.25%. Data on birthplace of grandparents showed a gradient with highest CCR5-delta32 frequencies from Eastern European Ashkenazim (~19.5% for those whose four grandparents come only from Russia, Poland, Hungary, Austria and Czechoslovakia; n = 197) which differs significantly from the frequency seen in Ashkenazi Jews from Western Europe (n = 101, p = 0.001). Homozygotes for CCR5-delta32 were genotyped with 3p21 region microsatellites. This has defined an ancestral haplotype on which the mutation first occurred and helped to date this event to between 40 and 50 generations ago or just over a thousand years ago. The population gradient, combined with the dating of the mutation by microsatellite allele frequencies, suggests an origin for the CCR5-delta32 allele in a population ancestral to the Ashkenazim. The distribution in non-Jewish populations in northern Europe has led others to postulate spread of the mutation by Vikings. It is hypothesised here that the link between the two populations could be the kingdom of Khazaria with subsequent admixture into both Swedish Vikings and Ashkenazi Jews. The basic driving force of evolution is through selection and the immune system has a role which, through the survival pressure exerted by viruses and other pathogens, has the potential to exert a great deal of selective force on the various components of this system. The effects of this pronounced selection on an immune system component can be seen for example in the increase of the CCR5-delta32 allele over the last thousand years to the current frequency. As mentioned, some immune system components are not affected by such straightforward selection. In the case of the TCRBV segments, effects on the immune repertoire can occur through MHC interaction at the point of thymic entry and in the effects of various superantigens, but the actual binding pockets that recognise antigen are themselves unable to be selected for (or against). The findings presented in this thesis provide support for the association of TCRBV gene segments with multiple sclerosis and also provide support for the further study of the role of the CCR5-delta32 allele in type 1 diabetes. Furthermore, data presented here suggests that the CCR5-delta32 allele had an origin in the Khazar Kingdom just over a thousand years ago, accounting for the allele frequencies in both the Ashkenazi Jews and in lands frequented by the Vikings. The definition of an extended ancestral haplotype for the CCR5-delta32 allele shows how the effect of selection of an allele of one gene can carry with it specific alleles of a large number of other genes as well.