No description available.

No description available.

The human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte (CTL) response is important in the control of HIV-1 infection. Due to the virus having a high rate of mutation, immune pressure can select for variants that are no longer recognised by CTLs to dominate the viral quasispecies. This is similar to how antiretroviral resistance emerges. HIV-1 is therefore adapting to both human leukocyte antigen (HLA)-restricted immune responses and antiretroviral therapy. This thesis initially focused on the natural CTL response to an HLA-B*51-restricted epitope in integrase. This HLA class I allele is associated with slow progression to AIDS; however, as no CTL-driven escape mutation has been fully defined within this integrase epitope, we cannot determine what contributes to the association of HLA-B*51 and natural control of infection. By longitudinally studying a cohort of early HIV-infected individuals, we observed the emergence of polymorphisms that abrogate a CTL response to this epitope. CTL escape may also prove to be the downfall of current immunotherapy strategies attempting to combat HIV infection. T cell receptors (TCRs) have been genetically modified to enhance their binding affinity to an HLA-A*02-peptide complex and transduced into CD8+ cells to create an HIV adoptive therapy. We demonstrate through in vitro selection pressure assays that escape from these cells may be a difficult task for the virus given that the TCR is able to recognise the majority of variants of this epitope. Antigen processing mutations may represent the only option for escape. How this may translate clinically will only be determined through in vivo studies, which must be meticulously monitored. Finally, when this high affinity TCR was fused to an anti-CD3 single chain variable fragment to create proteins capable of redirecting non-HIV-specific CTLs to HIV-infected cells, we found that the result was specific lysis. These proteins may supersede the use of TCR-transduced cells when used in synergy with antiretroviral therapy.

No description available.

No description available.

Cancer immunotherapy seeks to boost the host’s immune system to respond to tumor antigens. The adaptive immune system comprises of two arms, one that elicits a cellular immune response and one that elicits a humoral immune response. Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides presented to them in the context of class I major histocompatibility complex (MHC) molecules and are capable of killing tumor cells. CTL are educated to discriminate between foreign and self-antigen. Tumors frequently express self-antigen which usually makes them poorly immunogenic. Because tumors are genetically unstable, they may present excess self peptides and/or peptides in a reading frame different from wild type self proteins. These frameshift (FS) peptides, are caused by an insertion or deletion of nucleotides that disrupt translation of the normal reading frame and alters the protein produced such that it is non-self. Binding affinity, dissociation rate and the overall stability of the peptide/MHC/β₂-microglobulin complex are important considerations in determining the immunogenicity of a given peptide. Interaction between the anchor residues in a peptide and binding pockets in MHC are essential, but this interaction is not always strong enough to stimulate T cell responses. This indicates that not all amino acids of the peptide ligand bound to MHC are equally important for the functional outcome of the receptor engagement and that other amino acid residues in the sequence are important for binding. Optimized peptide ligands (OPL) are analogues derived from natural wild type antigenic peptides that contain amino acid substitutions at anchor and auxiliary residues. OPL can be rationally designed to generate a more robust immune response compared to that of the wild-type peptide. Active immunotherapy using OPL of tumor antigen epitopes are designed to elicit tumor-specific CTL that can overcome tolerance and either re-awaken or elicit new T-cell responses to an antigen. The work and principles presented here using brain tumor-derived peptides demonstrates that HLA-A*0201-restricted CTL generated against wild type, frameshift and OPL peptides elicit CTL that were able to recognize and respond to wild type, tumorderived peptides. The response was donor dependent in that not all individuals responded more strongly to OPL; a minority responded better to wild type peptide. This data further suggests that the rational design and testing of multiple peptides for the same epitope should elicit a broader response among different individuals than single peptide immunization.

Ovarian cancer, with the highest mortality rate amongst gynaecological malignancies in Australia, is the eighth most common cancer and the fifth cause of cancer-related deaths in women. Currently, five-year survival for women diagnosed with ovarian cancer is only 40 % and despite many patients experiencing remission, approximately 80 % of them will relapse due to residual micrometastasis. The limited impact of standard therapies on the prognosis for recurrent chemotherapy-resistant disease and the need to identify less toxic alternatives has motivated the development of strategies to combat the aggressive and life-threatening burden of ovarian cancer. A novel therapy against cancer utilises dendritic cells (DC), potent antigen presenting cells, to deliver tumour antigens to the immune system for the stimulation of cytotoxic T-lymphocyte (CTL) responses. DC immunotherapy has been used for the treatment of patients with ovarian cancer; however, clinical responses after the injection of antigen-loaded DC have been disappointing. Therefore, the identification of additional tumour associated antigens (TAA) is required. A TAA highly expressed in ovarian cancer cells, CA125, is a candidate target for DC-based immunotherapy. Initially, CTL responses to CA125 were studied in the context of HLA-A*0201. CD8+ T-cell responses specific for CA125 peptides (with high affinity for the MHC class I) were generated from cultures initiated with peptide-loaded monocyte-derived DC (Mo-DC). To expand the evaluation of T-cell recognition of CA125 to non-HLA-A*0201 individuals, messenger RNA (mRNA) was investigated as an antigen-loading vehicle. RNA encodes for the repertoire of epitopes presented by the TAA, potentially inducing immune responses in the context of multiple MHC class I and II molecules to known/unknown antigens. One focus of this study was to investigate a novel mRNA transfection system utilising mannan for the delivery of mRNA into DC. Initially the immunomodulating effect of mannan was examined in terms of DC activation. Mannan induced the phenotypic and functional maturation of immature Mo-DC in vitro. Next, the ability of oxidised mannan (OxM) linked to mRNA was investigated for its capacity to deliver enhanced green fluorescent protein (EGFP) mRNA into DC. We observed high transfection efficiencies in the murine and in human DC systems using low mRNA concentrations, in the absence of significant cell viability impairment. Interestingly, upon mRNA delivery via the OxM-PEI complex, DC maturation was induced to considerably higher levels as compared with that achieved with electroporation and non-transfected controls, this was measured by phenotype (CD83) and IL-12 secretion. Within this study, OxM-PEI did not deliver TAA encoding mRNA into DC for the stimulation of CTL. In summary, mannan is a novel strategy to deliver mRNA and a strong maturation signal simultaneously to human Mo-DC. The functional capacity of this system requires further investigation before it can be considered for clinical use. Electroporation has evolved as a superior method for mRNA delivery into DC as reported in the literature. Therefore, a comprehensive study was performed encompassing the critical issues associated with transfection efficiency, in order to standardise an electroporation protocol for use in DC immunotherapy schedules. EGFP was used as a model antigen to optimise mRNA uptake by Mo-DC by monitoring the expression of the reporter gene by FACS analysis. Influenza matrix protein 1 mRNA was, then, utilised as a model antigen for MHC class I restricted antigen presentation, for confirmation of the optimised loading parameters. The efficiency of this delivery system was assessed using CA125 mRNA in stimulating antigen-specific T-cell responses in PBMC of healthy individuals. CD4+ and CD8+ antigen-specific T-cell responses were generated recognising CA125 mRNA loaded Mo-DC and also ovarian cancer cell lines endogenously expressing CA125. This study has identified CA125 specific T-cell responses in healthy donors, allowing further investigation into the potential for its use as a candidate TAA in ovarian cancer immunotherapy. Furthermore, the use of Mo-DC transfected with mRNA encoding TAA is a promising strategy for the delivery of TAA in the generation of antigen-specific T-cell responses. In summary, the results gained from this PhD thesis should be taken into consideration when designing future DC immunotherapy strategies to combat one of the leading causes of cancer mortality in women, ovarian cancer.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 64-80).

Cytotoxic T Lymphocytes (CTL) are known to play an important role in clearing infectious virus from infected hosts in a variety of viral infections. Depending on the type of virus and mode of virus entry both class I and class II restricted CTL can contribute to protection from virus-induced disease. Although CD8 positive CTL are associated with virus elimination and control in many viral infections, elimination of neurotropic viruses from the Central Nervous system (CNS) is more complex due to the lowered expression of MHC antigens on neuronal cells. This failure to constitutively express high levels of MHC antigens by neurons could serve as an advantage to avoid damage to this differentiated and non-renewable tissue. However, abnormal induction of MHC antigens in the CNS mediated by CD4 positive lymphocytes or by astrocytes have also been shown to cause destructive inflammation in the CNS. The present study deals with CTL responses against one such neurotropic virus called Japanese Encephalitis Virus (JEV). JEV is a positive-stranded RNA virus that belongs to the flavivirus group, a group that is among the most important agents causing human encephalitis worldwide. Although passive transfer of monoclonal antibodies against this virus has been shown to confer protection of mice from lethal challenge with virus, neither the presence of CTL against this virus nor its role in conferring protection has been reported so far. Understanding the CTL responses against these viruses acquired importance in light of recent reports that neurovirulence of JEV and yellow fever viruses can be enhanced by the administration of virus specific antibodies. Hence this study was undertaken to examine the possibility of raising CTL specific to JEV. The specificity of the CTL raised, their therapeutic value and the ability of different lymphocyte subsets to mediate protection in vivo are dealt with in this study. Generation of CTL against JEV The generation of CTL against JEV in BALB/c mice, requires MHC defined cell lines that not only support virus infection but are also histocompatible. Several cell lines were initially examined for their ability to support JEV infection as a prc-rcquisitc before their utilization in in vivo and in vitro stimulation protocols aimed at generating JEV-specific CTL. Virus infection was monitored by immunofluorescence using JEV envelope-specific monoclonal antibodies as well as by titration of virus produced from infected cells by plaque assays. These different cell lines that were characterised for their ability to support JEV infection were then utilised to generate and monitor antiviral CTL. Several in vivo immunisation protocols were examined initially find out which of these infected cells prime BALB/c mice efficiently for generation of virus-specific CTL upon secondary stimulation in vitro with infected syngeneic cells. Immunisation of mice with infected cells per se was preferred over free virus since this was thought to facilitate priming against some viral non-structural proteins preferentially found on infected cells in addition to other viral structural proteins. It was observed that not only infected syngeneic and allogeneic cells but also infected xenogeneic cells prime BALB/c mice for the generation of JEV- specific CTL upon secondary restimulation in vitro. An optimal protocol was standardised for the generation of CTL against JEV. This included primary in vivo immunisation of mice followed by secondary in vitro restimulation of splenocytes with infected syngeneic cells. Either immunisation alone or in vitro stimulation of naive splenocytes alone was unsuccessful. The effector cells generated specifically lysed JEV-infccted P388D1 targets but not uninfected P388D1 or YAC-1 targets suggesting that the lysis on infected targets is not mediated by Natural Killer activity. Specificity and MHC restriction of anti JEV Effectors Cell depletion studies using complement mediated lysis were performed to examine the phenotype of the cells mediating virus specific lysis of infected targets. Depletion of Lyt 2.2+ or Thy 1+ but not L3T4+ sub-populations of effector cells inhibited lysis of infected targets showing that the effectors mediating virus-specific lysis were Lyt-2+ T cells. Examination of target specificities and MHC restriction of the antiviral CTL generated showed that although infected xenogeneic cells were used for immunisation, the effector cells recognised only infected syngeneic (P388D1, Sp2/0) and semisyngeneic (Neuro 2a, YAC-1) cells. Virus-specific recognition was found to be class I Kd and class I Dd restricted. These effector cells were also found to recognise cells infected with a closely related flavivirus, West Nile Virus (WNV) suggesting that they were crossreactive to some degree. Based on the consensus motif that has been established for H-2Kd associated peptides, several nonamers were predicted as possible CTL epitopes by scanning the deduced amino acid sequences of three strains of JEV and WNV. Among several predicted nonamers, three peptides were examined for their ability to reconstitute lysis of uninfected targets by polyclonal anti JEV CTL populations. Results demonstrate that peptides derived from NS1 and NS3 but not NS5 protein of JEV were able to partially reconstitute lysis of uninfected targets by effectors when pulsed with the appropriate peptide. Protective ability of the CTL raised against JEV To examine whether anti-JEV effectors raised in vitro could confer protection from intracerebral challenge with JEV, these effectors were adoptively transferred into adult BALB/c mice intracerebrally along with 10 x LDJ0 dose of JEV. More than 55% of these animals were protected from death and survived beyond 100 days after JEV challenge demonstrating that adoptively transferred anti-JEV effectors could indeed confer protection from lethal challenge with JEV. However, adoptive transfer of effectors by either intravenous or intraperitoneal routes did not protect adult mice from the lethal effects of intracerebral challenge with JEV. In contrast to adult mice, newborn mice were not protected from death by the adoptively transferred effector cells. This was also supported by experiments where a correlation was observed with the increasing age of mice and the success of protection conferred by the adoptively transferred effector cells. To establish the identity of cell subsets responsible for protection, Lyt 2, L3T4 or Thy 1 positive cells were specifically depleted from the polyclonal CTL by multiple cycles of complement mediated lysis and the remaining cells were adoptively transferred intracerebrally along with 10 x LD of JEV. These results demonstrate that both Lyt 2 and L3T4 positive T cells present in the effector population were necessary to confer protection of adult mice. Examination of virus-specific neutralising antibodies in the sera of protected and unprotected mice revealed that presence of L3T4 positive cells in the adoptively transferred population increases virus-specific neutralising antibodies. However presence of neutralising antibodies alone was not sufficient to confer protection. The protection required both Lyt-2 and L3T4 positive cells together. These studies could in the long term throw some light on similar observations about age dependant susceptibility to JEV in humans.

The present study has considered cellular and molecular requirements in T cell responses. The central role of T cell growth factors (TCGF) in T cell responses prompted us to study the regulatory events directing TCGF production in lectin stimulated cultures. It was found that normal spleen cells, activated with Concanavalin A for 24 h, develop suppressive cells that block de novo TCGF production by fresh spleen cells. The induction time for effector suppressor cells (nonadherent, Lyt-2-positive T cells) was found to be 18 h and to parallel the termination of TCGF production in situ. The suppressive mechanism is neither iji situ absorption of TCGF produced at control rates nor killing of TCGF producing cells. These results suggest that suppression of TCGF production is an active process which directly and reversibly blocks TCGF-producing cells. This study also indicated that ConA induced a very limited proliferation of Lyt-2- T helper cells (TH) in unselected T cell populations. The activation and growth requirements of Lyt-1+ TH cells were directly investigated and compared with those of Lyt-2+ cytotoxic T lymphocytes (CTL), as defined by the selective expression of Lyt differentiation antigens and functional activities. This analysis revealed a profound difference in activation and growth requirements between these T cell subsets. Thus, while Lyt-2+ CTL precursors can be induced to TCGF reactivity by soluble lectins, in the absence of specialized accessory cells,; Lyt-2" TH cell precursors show a strict accessory cell requirement both for activation and proliteration. Finally, the low level of TH cell effector function, detected in a primary responses to allo-MHC-antigens or lectins, appears to be due to the development of suppressive Lyt2+ T cells. The functional relevance of Lyt-2 antigens expressed on CTL membranes was further assessed in the last part of this study. Two distinct activation systems were used, namely MHC-antigens, provided as UV-irradiated stimulator cells or polyclonal induction by a 4 h pulse, with lectins. Both procedures were shown to selectively induce Lyt-2+ CTL precursors into TCGF reactivity without leading to mitosis, unless TCGF was added. In both cases it was found that monoclonal anti-Lyt-2 antibodies inhibited the two antigen- dependent phases of CTL responses namely, the initial induction step and target cytolysis. The analogy observed between antigen specific and lectin mediated indueton and target cytolysis, with regard to the susceptibility of inhibition by anti-Lyt-2 antibodies has lead to a general hypothesis on CTL activation. / <p>[4] s., s. 1-58: sammanfattning, s. 59-130, [12] s.: 6 uppsatser</p> / digitalisering@umu