Date of Award:

12-2013

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Animal, Dairy, and Veterinary Sciences

Committee Chair(s)

Christopher Davies

Committee

Christopher Davies

Committee

Gregory J. Podgorski

Committee

Kenneth L. White

Committee

Lee F. Rickords

Committee

Thomas D. Bunch

Committee

Dirk K. Vanderwall

Abstract

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.

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Dairy Science Commons

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