During viral infections, CD8+ T cells only respond to a select few epitopes derived from the respective foreign pathogen. These epitopes can be organized into a hierarchy, based on their ability to induce T cell priming. Such phenomenon is known as immunodominance. Cytotoxic T cells can be primed through the direct pathway, or the cross-priming pathway. The latter involves exogenously derived viral epitope presentation by uninfected professional antigen presenting cells. It has been previously reported that Lymphocytic Choriomeningitis nucleoprotein expressed in HEK cells (HEK-NP) could be cross presented to CD8+ T cells. In these studies we have used this same HEK-NP model to study the effects of LCMV-NP cross priming on the LCMV immunodominance hierarchy following viral challenge. Our results provide strong evidence that cross priming is an efficient route with which to induce cell-mediated immunity. We also highlight a regulatory role for cross priming in immunodominance by showing that a single dose of HEK-NP can completely shift the immunodominance hierarchy of a typical LCMV infection. Furthermore, we see that the induction of LCMV-NP cross priming boosts anti-viral immunity to subsequent LCMV infections. This work provides strong support for the physiological role that cross priming plays in normal cell-mediated immune responses. It may also provide relevant information to the realm of immunotherapy. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2007-07-10 14:33:18.115

Ankylosing spondylitis (AS) is associated with HLA-B*2704 and B*2705 but not with HLA-B*2706 and B*2709. Genome wide studies recently identified ERAP1 as an important genetic association in AS and could be the missing link in the pathogenesis of AS. I studied the implications of the two known actions of ERAP1 on AS pathogenesis. For assessing the peptide trimming function, surface HLA-B27 and MHC-I free heavy chain (FHC) expression on peripheral blood mononuclear cells of AS patients were studied. Subsequently, in an in vitro system of C1R cells expressing different AS-associated and AS-neutral HLA-B27 subtypes, I studied the effect of ERAP1 suppression on HLA-B27 and FHC expression. To assess the cytokine receptor shedding function, I studied serum cytokine receptor level variation with ERAP1 polymorphisms and its relationship to disease activity in AS patients. Finally, I studied the effect of variants of ERAP1 and other members of the antigen presentation machinery on radiographic severity in AS patients. AS patients with the major allele of the ERAP1 rs27044 polymorphism had higher FHC expression on monocytes. In C1R cells ERAP1 suppression led to an increase in intracellular FHC (IC-FHC) and B27-peptide complexes identified by a special MARB4 antibody, but only in C1R cells expressing the AS-associated subtypes HLA-B*2704 and B*2705. ERAP1 variants had no effect on serum cytokine receptor levels. Baseline radiographic severity was associated with ERAP1 polymorphism in univariate analysis only. LMP2 variants were associated with baseline radiographic severity in multivariate analysis. ERAP1 affects peptide presentation and FHC formation by HLA-B27 and could be the missing link in the pathogenesis of AS. ERAP1 through its differential HLA-B27 subtype interaction could explain why certain subtypes of HLA-B27 are associated with AS while others are not. Larger studies are required to look closely at the effect of ERAP1 on radiographic severity and progression in AS.

Ankylosing spondylitis (AS) is associated with HLA-B*2704 and B*2705 but not with HLA-B*2706 and B*2709. Genome wide studies recently identified ERAP1 as an important genetic association in AS and could be the missing link in the pathogenesis of AS. I studied the implications of the two known actions of ERAP1 on AS pathogenesis. For assessing the peptide trimming function, surface HLA-B27 and MHC-I free heavy chain (FHC) expression on peripheral blood mononuclear cells of AS patients were studied. Subsequently, in an in vitro system of C1R cells expressing different AS-associated and AS-neutral HLA-B27 subtypes, I studied the effect of ERAP1 suppression on HLA-B27 and FHC expression. To assess the cytokine receptor shedding function, I studied serum cytokine receptor level variation with ERAP1 polymorphisms and its relationship to disease activity in AS patients. Finally, I studied the effect of variants of ERAP1 and other members of the antigen presentation machinery on radiographic severity in AS patients. AS patients with the major allele of the ERAP1 rs27044 polymorphism had higher FHC expression on monocytes. In C1R cells ERAP1 suppression led to an increase in intracellular FHC (IC-FHC) and B27-peptide complexes identified by a special MARB4 antibody, but only in C1R cells expressing the AS-associated subtypes HLA-B*2704 and B*2705. ERAP1 variants had no effect on serum cytokine receptor levels. Baseline radiographic severity was associated with ERAP1 polymorphism in univariate analysis only. LMP2 variants were associated with baseline radiographic severity in multivariate analysis. ERAP1 affects peptide presentation and FHC formation by HLA-B27 and could be the missing link in the pathogenesis of AS. ERAP1 through its differential HLA-B27 subtype interaction could explain why certain subtypes of HLA-B27 are associated with AS while others are not. Larger studies are required to look closely at the effect of ERAP1 on radiographic severity and progression in AS.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 69-79).

<p>B lymphocytes are multifunctional and play important roles in both innate and adaptive immunity. The diverse roles of B cells can be attributed to the various and distinct types of B cells as determined by their origin, developmental stage, antigen specificity, and function.</p><p>Evidence suggests that human innate-like B cells (i.e., marginal zone and/or B1-like B cells) develop during fetal life. However, the characteristics of human fetal B-lineage cells are less understood. Recent studies of fetal and human umbilical cord B cells indicated that CD27, a well-established marker of human memory B cells, may also be expressed on human B1-like B cells. Indeed, CD27+ B cells are present in patients with hyper-IgM 1 (HIGM1) syndrome who are unable to generate GCs or memory B cells. In order to define the origin of naïve CD27+IgD+ human B cells, I studied B-cell development in both fetal and adult tissues.</p><p>In human fetal liver, most CD19+ cells co-express CD10, a marker of human developing B cells. Some CD19+CD10+ B cells express CD27, and these fetal CD27+ cells are present in the pro-B, pre-B, and immature/transitional B-cell compartments. Lower frequencies of phenotypically identical cells are also identified in adult bone marrow. CD27+ pro-B, pre-B, and immature/transitional B cells express recombination activating gene-1, terminal deoxynucleotidyl transferase, and Vpre-B mRNA comparable to their CD27− counterparts. CD27+ and CD27− developing B cells show similar immunoglobulin heavy chain gene usage with low levels of mutations, suggesting that CD27+ developing B cells are distinct from mutated memory B cells. Despite these similarities, CD27+ developing B cells differ from CD27− developing B cells by their increased expression of LIN28B, a transcription factor associated with the fetal lymphoid lineages of mice. Furthermore, CD27+ pro-B cells efficiently generate IgM+IgD+ immature/transitional B cells in vitro. Our observations suggest that CD27 expression during B-cell development identifies a physiologic state or lineage for human B-cell development distinct from the memory B-cell compartment.</p><p>Regarding B-cell repertoire, due to the random recombination of immunoglobulin V, D, and J gene segments during B-cell development, B cells are highly diversified in their antigen specificity. Through their specific B-cell antigen receptors (BCRs), B cells recognize foreign (and self-) antigens, and present these antigens to cognate T cells to elicit/establish humoral responses, such as germinal centers, immunological memory, and long-lasting circulating antibodies. Some bacteria and viruses escape the host’s immune system by mimicking host antigens, as B cells that recognize shared epitopes on self- and foreign antigens may provide protection against such pathogens; however, these B cells are normally eliminated by tolerance mechanisms during development. The extent of tolerization manifest among human B cells that recognize both self- and foreign antigens is unknown. Here, I and my colleagues use an efficient single B-cell culture method and multiplexed antigen-binding assays to determine the specificity of about 2,300 clonal IgG antibodies produced by the progeny of single transitional and mature B cells. We show that in healthy individuals, half of the self-reactive B cells crossreact with foreign antigen, and that the frequencies of crossreactive B cells decrease by half between the transitional and mature B-cell stages, indicating that a substantial fraction of foreign specificities is lost by the second tolerance mechanisms. In SLE patients, who show defective peripheral tolerance, frequencies of crossreactive B cells are unchanged between the B-cell stages. The crossreactive, mature B cells in SLE patients show distinct reactivity to foreign antigens. We propose that activating forbidden B cells may be a good strategy for protection against host-mimicking pathogens if we can control tolerance. </p><p>Activated B cells can present antigen to T cells, as well as differentiate into memory B cells and plasma cells. Indeed, activated B cells express high levels of MHCII and are considered to be professional antigen presenting cells (APC), along with dendritic cells and macrophages. APC can be used to discover the epitopes targeted in T-cell responses; T cells are co-cultured with autologous APC in the presence of antigens and T-cell responses are evaluated. With numerous epitope candidates, mapping T-cell epitopes requires large numbers of APC; the availability of APC in blood is a limiting component and leukapheresis is often required. Since B cells can be expanded in vitro more easily than other APC, they represent a solution for the challenge of isolating adequate numbers of APC from blood in order to determine T-cell antigen specificity. I modified our single B-cell culture to support efficient activation and proliferation of both naïve and memory human B cells for the purpose of generating large numbers of autologous APC. Briefly, naïve or memory B cells recovered from blood are cultured with recombinant human IL-2, IL-4, IL-21, and BAFF on CD154+ feeder cells; this culture supports extensive B-cell proliferation, with approximately 103 fold increases following 8 days in culture, and 106 fold increases when cultures are split and cultured for 8 more days. The capacity for continued proliferation is stable for at least another week. In culture, a significant fraction of naïve B cells undergo isotype switching and terminally differentiate into plasmacytes. Culture-derived (CD) B cells are readily cryopreserved, and when recovered, retain their ability to proliferate and differentiate. Significantly, proliferating CD B cells express high levels of MHCII, CD80, and CD86. I have examined the APC function of CD B cells and found that they present both allo- and microbial antigens to autologous T cells with comparable efficiency to PBMC. Moreover, I am able to activate and expand antigen-specific memory B cells; these cultured cells are highly effective in presenting antigen to T cells. This culture method provides a platform for studying the BCR and TCR repertoires within a single individual.</p> / Dissertation

Salmonella enterica serovar Typhimurium (ST) is an intracellular bacterium that resides within the phagosome of infected cells. ST is the causative agent of gastroenteritis in humans and typhoid like disease in mice. ST infects epithelial cells and phagocytic cells such as dendritic cells (DCs), which are immune sentinels that have been regarded as the most critical antigen-presenting cell (APC). I evaluated the role of CD8α DCs, a subset of DCs capable of antigen presentation of phagosomal pathogens to activate CD8+ T cells. Furthermore, I assessed the role of key cytokines such as the group of classical anti-viral cytokines known as Interferon-I (IFN-I), on licensing CD8+ T cells. Interestingly, IFN-I signalling was necessary for production of inflammatory cytokines and induction of cell death, which activated CD8+ T cells and clearance of ST. Lastly, I examined the role of key cell death pathways in innate immune protection against ST. In particular, I addressed how signalling pathways in necroptosis and pyroptosis are critical for the production of IL-1beta and IL-18 which mediate immune protection against ST. Determining the mechanisms of which DCs engage innate and adaptive immune responses against phagosomal bacteria is the central question of my study and is addressed by examining critical roles of DC function, inflammation and cell death.

The major histocompatibility complex class I (MHC-I) antigen presentation pathway is necessary for the immune system to be able to detect, control, and eliminate cancers. MHC-I binds oligopeptides derived from cellular proteins and presents them on the cell surface to CD8+ T cells. Consequently, the CD8+ T cells can monitor whether any cells are making abnormal proteins and, if so, can destroy those cells. Because MHC-I presentation is not essential for cell viability, immune selection pressure often leads to cancers that are MHC-I low as they can better evade CD8+ T cell recognition. It is, therefore, important to fully understand the mechanisms of MHC-I presentation as this will identify new ways to target and exploit the pathway for cancer therapeutics. Although several components of the MHC-I pathway have already been characterized, some knowledge gaps remain. Unbiased forward genetic screens from our lab identified some novel gene candidates, such as IRF2, which positively regulate MHC-I presentation. In this dissertation, I will reveal which antigen presentation pathway genes are transcriptionally controlled by IRF2 and contribute to the MHC-I presentation deficiency observed in cells lacking IRF2 and I will also show that IRF2 negatively regulates PD-L1 expression. By influencing both MHC-I antigen presentation and PD-L1 expression in this manner, cancers lacking IRF2 (of which there are many) are both harder to see and more difficult to eliminate.

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