Doctor of Philosophy / Department of Biology / Stephen Chapes / Antibodies are an essential part of the immune system. Each B cell, a type of white blood cell, produces a unique antibody. This antibody molecule is comprised of two identical light chains and two identical heavy chains. Each chain has a variable region, which is responsible for antigen binding, and a constant region, which is responsible for effector function in the host. The variable region in the heavy chain is composed of three gene segments, the variable (V), diversity (D), and joining (J) gene segments. The light chain is composed of only V- and J-gene segments. Each immunoglobulin locus contains multiple versions of each gene segment, ranging from over 130 possible V gene segments in the heavy chain to four possible J-gene segments in both the heavy and kappa light chain. The recombination of gene segments occurs in the germline DNA and results in the formation of the unique antibody. The diversity and binding abilities of the antibodies are important for a proper and robust immunological response. Of importance to binding and specificity is the complementary determining region three (CDR3) which plays a major role in determining specificity and antibody-antigen binding. Due to its uniqueness, is used as a measure of diversity in the repertoire.
In this work, I used Illumina MiSeq 2x300nt high-throughput sequencing to assess the mouse splenic transcriptome. The work I present here shows the splenic immunoglobulin gene repertoire from unchallenged, unvaccinated conventionally housed mice, mice flown aboard the International Space Station (ISS), and mice challenged with tetanus toxoid (TT) and/or adjuvant (CpG) and subjected to skeletal unloading by antiorthostatic suspension (AOS). AOS is used to induce some of the physiological changes that parallel those that occur during space flight. The characterization of the repertoire includes analysis of V-, D-, and J-gene segment usage, constant region usage, V- and J-gene segment pairing, and CDR3 length and usage.
The work included validation of the methodology needed for tissue preparation and storage aboard the ISS, showing that the data obtained was similar to those used in standard ground-based methodologies (Chapter 2). I further validated our nonamplified sequencing methodology with comparisons to methods that use amplification as part of the process (Chapter 3). My work characterized the antibody repertoire of the conventionally housed C57BL/6J mouse (Chapter 4), an important mouse strain in the field of immunology, and demonstrated the homogeneity of gene segment usage in unchallenged animals. We also demonstrated that short duration (~21 days) space flight does not significantly alter the antibody repertoire (Chapter 5). The work culminates in an AOS study to assess changes to the B-cell immunoglobulin repertoire after vaccination with TT and/or CpG. The results show that changes to V-, D-, and J-gene segment usage occur after antigen challenge with AOS causing decreased class switching and frequency of plasma cells. Tetanus toxoid challenge decreased multiple gene segment usage and CpG administration increased isotype switching to the IgA constant region (Chapter 6).
| Identifer | oai:union.ndltd.org:KSU/oai:krex.k-state.edu:2097/38796 |
| Date | January 1900 |
| Creators | Rettig, Trisha Ann |
| Publisher | Kansas State University |
| Source Sets | K-State Research Exchange |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation |
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