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Author ORCID Identifier

https://orcid.org/0000-0002-0459-3945

AccessType

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Animal Biotechnology & Biomedical Sciences

Year Degree Awarded

2021

Month Degree Awarded

February

First Advisor

Cynthia L Baldwin

Subject Categories

Immunology and Infectious Disease

Abstract

CAPRINE gd T CELL BIOLOGY

AUGUST 2020

ALEHEGNE W YIRSAW, DVM, ADDIS ABABA UNIVERSITY, ETHIOPIA

MSC., ADDIS ABABA UNIVERSITY, ETHIOPIA

PHD., UNIVERSITY OF MASSACHUSETTS AMHERST

DIRECTED BY: PROFESSOR CYNTHIA L BALDWIN

Goats are important food animals and are disseminated globally. Their productivity is impacted by infectious diseases thus impacting the livestock owners and the community. γδ T lymphocytes or T cells have vital roles in immune responses in mammals and thus we identified information gaps regarding these cells in goats. We used genome annotation, PCR amplification of genomic DNA and cDNA, and Sanger and PacBio sequencing for this research to evaluate the important receptors of these cells, i.e. the T cell receptor (TCR) and the hybrid pathogen pattern recognition receptor (PRR) and TCR co-receptor known as WC1. The annotated TCRγ (TRG) and TCRδ (TRD) loci were similarly organized in goats as previously reported for cattle and the gene sequences were highly conserved between these ruminant species. However, the number of genes varied slightly as a result of duplications within loci and differences occurred in mutations resulting in pseudogenes. Goats have 6 functional and 1 pseudogene TRG gene cassettes and TRD genes have one constant gene (TRDC), 4 joining genes (TRDJ), 7 diverging genes (TRDD and 34 TRD variable genes (TRDV). In cattle, the WC1+ γδ T cells only use TCRγ genes from one of the 6 available cassettes and we found the same conservation for goat WC1+ γδ TCRγ gene usage. With regard to the co-receptor, we found evidence for up to 30 caprine WC1 genes, twice that of cattle, and seven different WC1 gene structures of which 4 are unique to goats. The most distal WC1 scavenger receptor cysteine-rich (SRCR) domains known as SRCR a1, based on sequence and position, were highly conserved among goat breeds but fewer were conserved between goats and cattle. Caprine WC1 genes also had multiple splice variants of transcripts coding for the intracytoplasmic domains that in some cases eliminated tyrosines shown previously to be important for signal transduction. In summary, goat WC1 molecules may have expanded functions given their expanded number and structure variations. Using immunofluorescence and flow cytometric analysis and monoclonal antibody (mAb) GB21A, reactive with the TCR δ constant region (TRDC), we found that the proportion of gd T cells in caprine blood was not significantly less than that of CD4 or CD8 T cells, with WC1+ γδ T cells ranging from ~20-90% of the total. Less than half of the WC1+ cells could be classified as WC1.1+ or WC1.2+ subpopulations by additional mAb staining indicating a large third subpopulation in goats. Since WC1 gene expression may direct pathogen responses the WC1 genes expressed by subpopulations of WC1+ γδ T cells were also identified by next-generation sequencing. To experiments to evaluate responses to pathogens, we found that naïve gd T cells proliferated in in vitro recall cultures stimulated with Leptospira borgpetersenii or Mycobacterium avium paratuberculosis (MAP) above that in control cultures without antigen or those with M. bovis BCG. The responding cells included both WC1+ and WC1- γδ T cells and the WC1- γδ T cells produced IL-17. In ex vivo PMA-stimulated cultures WC1+ γδ T cells were shown to also produce IL-17 while WC1- γδ T cells produced both IL-17 and interferon-g (IFNγ). In the future, more extensive studies of caprine gd T cells will be needed to evaluate the role of the various γδ T cell subpopulations to important livestock pathogens. Knowing this information will be important for understanding how to engage these cells in vaccine designs.

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