Thesis (M.S.)--Villanova University, 2008. / Biology Dept. Includes bibliographical references.

Thesis (Ph. D.)--University of Kentucky, 2000. / Title from document title page. Document formatted into pages; contains vi, 137 p. : ill. Includes abstract. Includes bibliographical references (p. 108-135).

CD4+ lymphocytes (T helper cells) play a crucial role in orchestrating adaptive immune responses. This project aims to characterise the CD4+ cells in zebrafish (Danio rerio) for a better understanding of adaptive immunity in teleost fish. I cloned three CD4 homologues, termed zfCD4 and zfCD4 related (zfCD4rel1 and zfCD4rel2), with both reported previously in other bony fish species. The zfCD4 and zfCD4rels transcripts were detected in immune organs in zebrafish and were most highly expressed in the lymphocyte population. A moderate induction of the zfCD4 and zfCD4rels (and other immune related genes) was seen in kidney, spleen and intestine after poly (I:C) injection. Two antibodies against CD4 and IFN-γ, respectively, have been validated using various immunostaining approaches for further functional studies. The CD4 positive cells ranged from 20-30% of lymphocytes in zebrafish, similar to what is seen in other vertebrates. The expression level of IFN-γ and other Th cell related genes were analysed in immunised fish following re-stimulation with antigen, revealing that in zfCD4+ lymphocytes an increased expression of cytokines and master transcript factors was seen when the same antigen was used for boosting. This is the first demonstration of Th-type responses effected by CD4+ lymphocytes in a poikilotherm. Lastly, I studied an aspect of Treg function in zebrafish, focused on a master transcription factor of Tregs, the FoxP3 gene. Knocking down FoxP3 genes in zebrafish resulted in modulated gene expression of cytokines and transcription factors associated with Th and Treg cells, providing some evidence that the immune tolerance function of Treg cells may exist in teleost fish, with some sub-functionalisation of the two FoxP3 paralogues apparent. This thesis extends our knowledge into teleost adaptive immune responses mediated by CD4+ Th cells and putative FoxP3+ Treg cells and may aid future studies using zebrafish as a model of vertebrate immune function.

CD4 is a co-receptor for binding of T cells to antigen-presenting cells (APC) and the primary receptor for human immunodeficiency virus-I. The disulfide bond in the second extracellular domain (D2) of CD4 is reduced on the cell surface, which leads to formation of disulfide-linked homodimers. A large conformational change must take place in D2 to allow for formation of the disulfide-linked dimer. Domain swapping of D2 is the most likely candidate for the conformational change leading to formation of two disulfide-bonds between Cysl30 in one monomer and Cysl59 in the other one (Cys133 and Cysl62 in the mouse CD4). Thus, we hypothesized that the domain swapping of D2 in CD4 regulates its co-receptor function of antigenspecific T cell activation. We found that mild reduction of the extracellular part of human CD4 resulted in formation of disulfide-linked dimers. We then tested the functional significance of dimer formation for co-receptor function using the engineered Jurkat T cell system by expressing wild-type or disulfide-bond mutant mouse CD4. Eliminating the D2 disulfide bond markedly impaired CD4's coreceptor function as assessed by antigen-specific IL-2 production. Exogenous wild type thioredoxin, but not redox-inactive thioredoxin, could inhibit the CD4-mediated IL-2 production, suggesting that the redox state of D2 disulfide bond is controlled by this oxidoreductase. Furthermore, structural modeling of the complex ofthe T cell receptor and domain-swapped CD4 dimer bound to class II major histocompatibility complex and antigen supports the domain-swapped dimer as the immune co-receptor. The known involvement of D4 residues Lys318 and Gln344 in dimer formation isalso accommodated by this model. These findings imply that disulfide-linked dimeric CD4 is the preferred functional co-receptor for binding to APC. Strategies to promote dimerisation of CD4 should, therefore, enhance the immune response, while inhibiting dimer formation is predicted to be immunosuppressive.

Zugl.: Leipzig, Univ., Diss., 2007

published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

No description available.

Abstract not available

Title from PDF of title page (University of Missouri--Columbia, viewed on March 10, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Thesis advisor: Dr. Xudong Fan. Includes bibliographical references.

Here we report the expression and purification of two recombinant Fasciola hepatica enzymes, catL2 and catL5 which were used to perform cleavage studies with substrates potentially encountered by the parasite in vivo; BSA, hIgG3K and the important T cell marker, CD4. We examined the digestion products generated by the cleavage of human CD4 with catL5 using mass spectrometry and predicted candidate cleavage sites by performing a theoretical digest of the protein. / Ovine CD4 is also of interest to us as a target of F. hepatica cathepsin L activity. Here we confirm a recently reported ovine CD4 cDNA sequence and the existence of a single nucleotide polymorphism (T/C) within this sequence. The polymorphism translates to a serine-proline switch near the hinge region of the protein. Additionally, we have found that this polymorphism is also present in genomic DNA, suggesting that two alleles of CD4 exist in the ovine genome.