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Immune evasion is an emerging hallmark of cancer. Dysregulation of the major histocompatibility complex class I (MHC-I) is a frequent mechanism of immune evasion utilized by tumor cells and is particularly relevant to the pathogenesis of B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). A better understanding of MHC-I dysregulation in B-cell lymphomas is necessary to identify factors related to the risk, development, and progression of these tumors. In this thesis, we investigate the role of MHC-I dysregulation in DLBCL and cHL through the application of computational approaches to study genomic data. First, we introduce some background information about the normal function of MHC-I in the immune response to cancer and viral infection as well as the phenomenon of MHC-I dysregulation in the context of cancer. We provide an overview of how factors such as germline zygosity of HLA class I (HLA-I) genes and somatic alterations in the genes B2M and HLA-I that encode the protein subunits of MHC-I contribute to the development of DLBCL and cHL. Second, we present a study of the effects of HLA-I allele zygosity on survival in a cohort of 519 DLBCL patients treated with R-CHOP immunochemotherapy stratified by molecular subtype. Homozygosity in HLA-I was associated with a worse overall survival in patients whose tumors were classified in the “EZB” subtype, associated with somatic mutation in the epigenetic regulator EZH2. We find an association between the zygosity of the genes HLA-B and -C specifically and overall survival in EZB-DLBCL. These findings indicate that HLA-I zygosity may be a risk factor for worse clinical prognosis in patients with the EZB subtype of DLBCL. Third, we present a study of the genetic landscape of cHL tumors that are associated with infection with Epstein-Barr virus (EBV). We analyze inherited HLA-I allele types, somatic mutations, copy number changes, and mutational signatures in a cohort of 57 cHL patients (15 EBV-positive). We find that EBV-positive cHL is genetically distinct from EBV-negative cHL and is characterized by lower somatic mutation load and different activities of mutation signatures. Further, we find that cHL tumors are characterized by different patterns of MHC-I dysregulation depending on the EBV infection status. Germline homozygosity in HLA-I was associated with the EBV-positive subtype of cHL, while somatic alterations in HLA-I were associated with the EBV-negative subtype of cHL. These results suggest that inherited HLA-I homozygosity may be a risk factor for the EBV-positive subtype of cHL. Fourth, we expand our study of HLA-I in virus-associated cHL to perform a comparative analysis of virus-positive and virus-negative tumors in nine cancers linked to five viruses. We find that virus-positive tumors occur more frequently in males and show geographical disparities in incidence. Genomic analysis of 1,658 tumors reveals virus-positive tumors exhibit distinct mutation signatures, recurrent mutations in the RNA helicases DDX3X and EIF4A1, and a lower somatic mutation burden compared to virus-negative tumors of the same cancer type. We find that germline homozygosity in HLA-I is a potential risk factor for the development of EBV-positive cHL, but not other common virus-associated solid or hematological malignancies. Finally, we present in the Appendix a study of the genomic characterization of plasmablastic lymphoma (PBL), a rare EBV-associated B-cell lymphoma that occurs in the context of immunodeficiency caused by human immunodeficiency virus (HIV) infection. We find that PBL is characterized by mutations leading to constitutive activation of the JAK-STAT pathway. We additionally identify recurrent mutations in immune-related genes, such as B2M. These findings indicate a potential role for MHC-I and immune dysregulation in the pathogenesis of other B-cell lymphomas.

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Long non-coding RNAs (lncRNAs) have been implicated as important regulators of inflammation through various mechanisms in both the innate and adaptive immune systems of mice and humans. The majority of SNPs identified by GWAS to be associated with autoimmune disorders lie within non-coding areas of the genome, including genes for lncRNAs. To identify lncRNAs with relevancy to inflammation and autoimmunity, a discovery pipeline was used to find lncRNAs differentially expressed in TLR4 activated murine macrophages, conserved between mice and humans, and harboring GWAS identified SNPs associated with autoimmune disorders. Two of the six candidate lncRNAs identified, Lnc15 and Lnc13, are decreased in activated macrophages and are associated with both celiac and Crohn’s disease. To further explore the regulation and influence of these two lncRNAs during inflammation and its resolution, a variety of in vitro and in vivo techniques were utilized, including novel mouse knockout models. An investigation of Lnc15 was conducted in cells of both the innate and adaptive immune system, where the dominant isoform of Lnc15 was identified to be a ~1.4 kb transcript localized to the cytoplasm in both murine macrophages and T cells. Analysis of Lnc15 regulation was conducted in activated murine macrophages, focused on TLR signaling. Through stimulating macrophages with specific TLR ligands, Lnc15 was found to be decreased by TLR2, TLR3, and TLR4 signaling, likely dependent upon both MYD88 and TRIF. While not dependent upon NF-κB, protein synthesis is required for TLR induced decreases in Lnc15 levels. Conversely, activated neutrophils significantly increase Lnc15 levels, although the mechanism of regulation is not yet known. Mice lacking Lnc15 globally were found to be more susceptible to DSS induced colitis, which is likely dependent upon a defect in the innate immune system. In the adaptive immune system, Lnc15 was found to be specifically upregulated in Tregs compared to other T cell subsets. Lnc15 deficient Tregs had a reduced suppressive capacity in vitro, but not in vivo in a T cell induced model of colitis. These findings suggest Lnc15 plays a role in Treg suppressive capacity under certain conditions, but the exact mechanism influenced remains to be identified. Additionally, overexpression of Lnc15 in a murine T cell line resulted in a decrease in Rorc expression. A Lnc15 RNA pulldown experiment identified USF2, a transcription factor known to regulate Rorc expression, and UBR5, a ubiquitin-protein ligase known to influence RORyt stability, as protein interactors of Lnc15. These data indicate that Lnc15 can influence aspects of RORyt biology, which implicates Lnc15 as a regulator of either the plasticity between Tregs and Th17 cells, or Treg ability to suppress inflammatory Th17 cells. An investigation into Lnc13 regulation by disease relevant cytokines was conducted with a series of macrophage stimulation experiments. Lnc13 was found to be positively regulated by cytokines with an anti-inflammatory capacity, including IL-6, IL-4 and IL-10. When Lnc13 deficient macrophages were polarized, a higher expression of Il6 was detected in both M1 and M2 macrophages, suggesting a regulatory connection between Lnc13 and IL-6 across macrophage activation states. Previously identified Lnc13 target genes displayed a quicker transcriptional response to LPS stimulation in Lnc13 deficient macrophages. Additionally, when the Lnc13 mouse was crossed with the DQ8 transgenic mouse model and challenged to gluten, the ileum tissue of Lnc13 deficient mice expressed higher amounts of Il12 and Ifng, cytokines directly relevant to celiac disease. These findings provide support for Lnc13 as a novel regulator of macrophage response and cytokine expression in response to disease relevant stimuli.

During tumor growth there are a series of phenotypic and functional changes that occur in macrophages (M_Φ) that ultimately lead to the immunosuppression of the tumor-bearing host (TBH). To investigate the phenotypic changes of M_Φ during tumor growth, we examined the expression of the M_Φ surface antigens, Mac-1, Mac-2, Mac-3, and Ia on peritoneal and splenic M_Φ. In the peritoneal cavity there was no change in the percentage of Mac-1⁺ M_Φ but a decrease in the percentage of Mac-2⁺, -3⁺, and Ia⁺ M_Φ during tumor growth. In addition, three distinctly sized populations of peritoneal M_Φ, showing differential antigen expression, also shifted during tumor growth. In the peritoneal cavity there was a decrease in the percentage of M_Φ co-expressing the Mac-2, -3, and Ia antigens, leading to a shift towards Mac-1⁺ 2⁻ 3⁻ Ia⁻ TBH M_Φ. In splenic M_Φ, the percentage of Mac-1⁺, -2⁺, and -3⁺ M_Φ increased, while the percentage of Ia⁺ M_Φ decreased. Splenic M_Φ showed an increase in the percentage of M_Φ co-expressing Mac-1, -2, and -3 antigens and a decrease in the percentage of M_Φ co-expressing Ia, leading to a shift towards a Mac-1⁺ 2⁺ 3⁺ Ia⁻ TBH M_Φ. Taken together, these data suggest that tumor growth alters the phenotype of M_Φ and causes a shift in M_Φ subpopulations. After measuring the phenotypic changes in M_Φ during tumor growth, changes in M_Φ accessory function to T cells were assessed. TBH M_Φ have significantly reduced accessory activity for autoreactive T cells. This reduction is caused by decreased Ia antigen expression and increased production of the suppressor molecule, prostaglandin (PG). TBH M_Φ down-regulated autoreactive T cell responsiveness to interleukin (IL)-1, IL-2, and IL-4. In addition to TBH M_Φ reducing T cell responsiveness to cytokines, TBH CD4⁺ T cells alone were less responsive to the cytokines IL-1, IL-2, and IL-4. To examine the responsiveness of M_Φ to activation signals, lipopolysaccharide (LPS) was incubated with normal and TBH splenic M_Φ and assessed for their phenotypic, functional, and cell-cycle changes. The data showed that TBH M_Φ had a decreased responsiveness to LPS. We showed that there was a shift from an Ia⁺ M_Φ in the normal host to an Ia⁻ M_Φ in the TBH. Concomitant with the shift in TBH M_Φ Ia⁻ phenotype was a change in TBH M_Φ function. Normal and TBH Ia⁻ M_Φ were suppressor M_Φ. TBH Ia⁻ M_Φ, however, suppressed autoreactive and alloreactive CD4⁺ T cells significantly more than could their normal counterparts. Tumor growth causes quantitative and qualitative changes in Ia⁻ suppressor M_Φ. Although Ia⁻ M_Φ-mediated suppression seemed to be the major source of down-regulation of CD4⁺ T cells, CD8⁺ T cells were not without fault. In the TBH, there was an increase in the percentage of CD8⁺ T cells and an increase in CD8⁺ T cell-mediated suppression. In conclusion, tumor growth leads to a change in immunoregulation that causes suppression of the immune response. / Ph. D.