My dissertation hypothesis is that bovine trophoblast cells express cell-surface and secreted non-classical major histocompatibility complex class I (MHC-Ib) proteins which inhibit NK cells and other leukocytes by binding to inhibitory receptors (e.g., LILRB1, LILRB2, KIR2DL4, and/or CD94/NKG2A). Extremely polymorphic and ubiquitously expressed classical MHC class I (MHC-Ia) proteins, which present foreign antigenic peptides to CD8+ T lymphocytes, are involved in acceptance or rejection of tissue grafts. Non-classical MHC class I (MHC-Ib) glycoproteins, such as Human Leukocyte Antigen-G (HLA-G) and murine Qa-2, are important modulators of the maternal immune system during pregnancy. MHC-Ib proteins are: (a) oligomorphic or monomorphic, (b) expressed in specific tissues under specific condtions, and (c) produced as surface and/or soluble isoforms due to alternative splicing. Third trimester-bovine trophoblast cells express both MHC-Ia and MHC-Ib proteins. The MHC-Ib proteins expressed by trophoblast cells during the third trimester of pregnancy are encoded by four bovine leukocyte antigen (BoLA) loci: BoLA-NC1, BoLA-NC2, BoLA-NC3, and BoLA-NC4. Two MHC-Ia (N*01701 and N*01802) and three MHC-Ib (NC1*00501, NC3*00101 and NC4*00201) proteins showed cell-surface expression in transfection studies performed in murine P815 and human K562 cells. Two additional isoforms, NC1*00401 and NC2*00102, were not detected on the surface of these cells. Nevertheless, both class Ia proteins, N*01701 and N*01802, and five class Ib proteins, NC1*00401, NC1*00501, NC2*00102, NC3*00101, and NC4*00201, were detected in crude cell lysates on Western blots. Precipitation of proteins from culture supernatants showed that cell-surface MHC-Ia (N*01701 and N*01802) and MHC-Ib proteins (NC1*00501, NC3*00101, and NC4*00201) are shed from the surface of these cells into the media. The mechanism of shedding of these proteins is, however, not known. Monoclonal antibodies W6/32, IL-A88, H1A, H6A, H11A, H58A, and PT-85A recognized surface MHC-I isoforms with varying affinity. We were able to develop a sandwich enzyme-linked immunosorbent assay (ELISA) using either H1A or IL-A88 antibody as the capture antibody and the W6/32 antibody for detection. We produced monoclonal antibodies against cattle NC1*00501 and NC3*00101 proteins. One monoclonal antibody generated against BoLA-NC3*00101 was highly specific. Unfortunately, due to failure to clone the NC3*00101- hybridoma, we no longer have an infinite source of this monoclonal antibody for NC3*00101. We eluted peptides from NC3*00101-transfected MHC-null K562 cells and identified peptides using liquid chromatography-mass spectrum (LC-MS) analysis. Analysis of peptide binding data using the SAS Proc mixed statistical program, suggested that the peptide EVTNQLVVL is a potential peptide ligand, which can be used to make tetramers for enumeration of antigen-specific leukocytes.

The precise regulation of Major Histocompatibility class II (MHC-II) genes plays an important role in the control of the adaptive immune response. MHC-II genes are expressed constitutively in only a few cell types, but their expression can be induced by the inflammatory response cytokine interferon gamma (INF-γ). The regulation of MHC-II is controlled by a Master Regulator, the class II transactivator (CIITA). Multiple studies have shown that CIITA regulated expression of MHC-II is controlled and induced by INF-γ. It has been also shown that a functional CIITA gene is necessary for the expression of MHC-II genes. CIITA is thus a general regulator of both constitutive and inducible MHC-II expression. Although much is known about the transcription factors necessary for CIITA expression, there is little information as to the epigenetic modifications and the requisite enzymes needed to provide these transcription factors access to DNA. Previous studies in the Greer lab have shown that increased levels of acetylation of histones H3 upon INF-γ stimulation, as does tri-methylation of H3K4 upon prolonged cytokine stimulation. Similar observations were made at early time points post IFN-γ stimulation, where there is an instantaneous increase in the levels of H3K18ac and H3K4me3. In contrast to this, the levels of silencing modifications begin to drop with in the first 20 minutes of IFN-γ stimulation. The binding of STAT1 reaches its peak at about 60 minutes and the first transcripts for the protein start to appear as early as 40 minutes post the cytokines stimulation. Our study is the first to link the rapidly occurring epigenetic changes at the CIITA promoter pIV to EZH2

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 106-125).

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 89-102).

As individuals and populations diverge ecologically, they become exposed to new parasites and pathogens with potentially harmful fitness consequences. Populations are therefore expected to evolve resistance, possibly at a cost of less resistance to parasites rarely encountered parasites. This trade-off in resistance should generate local adaptation to parasites in different habitats. In chapter one, I show how local adaptation can potentially evolve in response to variation in parasite exposure among eighteen ecologically variable populations of threespine stickleback (Gasterosteus aculeatus). Within populations infection appeared to reflect morphology/diet based exposure differences among individuals. Among populations, however, these patterns were absent or reversed, consistent with the evolution of local adaptation. In chapters two and three I set out to test whether variation major histocompatibility (MHC) genes can underly such local adaptation in stickleback. MHC genes are important components of vertebrate immunity; however, there is little direct empirical support for spatially divergent selection driving local adaptation on MHC loci in the wild. In chapter two I tested for the action of parasite mediated balancing and divergent selection on on MHC loci using naturally infected stickleback in three replicate lake-stream pairs. Despite consistent divergence in parasites and MHC alleles, lakes tended to show decreased parasite burdens with increased allelic richness (consistent with balancing selection), while streams showed some support for divergent selection between lake and stream types. In chapter three I use the same lake-stream pairs to investigate how divergent selection could instead be reflected in variation in the effects of individual MHC alleles among populations. When comparing parapatric populations experiencing gene flow, MHC alleles maintained at relatively high frequency in one population were more likely to be associated with reduced, rather than increased, parasite abundances in that population. Allopatric populations experiencing no gene flow showed no such general relationship between allele frequency and resistance. These results are only consistent with spatially divergent selection, and imply that gene flow and environmental heterogeneity can be important for maintaining MHC diversity. / text

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 147 pages. Includes vita. Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.