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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 regulation of the immune system is complex, with many factors involved in controlling immune cell development, activation and homeostasis. These factors include neuropeptides as well as classic immunoregulatory molecules such as cytokines, chemokines and hormones. Neuropeptides and tachykinins in particular are known to be involved in immune response modulation through a cascade of events including vasodilation, plasma extravasation, the activation of immune cells, the secretion of pro-inflammatory cytokines and the recruitment of more immune cells. Furthermore, there is growing evidence that tachykinins play a role in hematopoiesis with Substance P as the proposed effector molecule. In 2000, our lab discovered a new tachykinin with remarkable structural similarity to SP and SP-like neurokinin receptor binding affinity. This molecule was designated Hemokinin-1 due to its expression in hematopoietic cells and its function in B cell development. Further gene expression analysis of HK-1 reveals a wide expression pattern although HK-1 transcripts are found predominantly in peripheral tissues while SP is mainly expressed in neuronal tissue. Based on this differential expression pattern, it has been suggested that HK-1 may act as the peripheral tachykinin and may have functions distinct from SP. In addition, given the crossreactivity of the SP antibodies to HK-1, it is important to determine whether HK-1 is the actual mediator of some functions previously attributed to SP. In this thesis, we examine the differential expression pattern of HK-1 to determine molecular mechanisms of regulation of HK-1 transcription and ultimately provide clues to its function in the immune system. In our analysis of the HK-1 promoter, we found a major difference in the basic transcriptional control of HK-1 and SP at the level of transcription initiation and identified several transcription factors including CREB and NFκB involved in regulating TAC4 gene expression in immune cells. Data presented in this thesis also reveal that the HK-1 gene is a direct target of Early B-cell Factor, a transcription factor known to activate B cell-specific genes as well as genes involved in adipogenesis and neuronal development. Our results show EBF regulates HK-1 gene expression in differentiating B cells as well as a monocytic cell line. Our data indicate EBF may also be responsible for the high levels of HK-1 transcript in the olfactory epithelium, suggesting a bridge between the nervous system and the immune system.