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There is currently a need for vaccine adjuvants that are effective for eliciting Th1-type CD4 and CD8 T cell responses when formulated with protein and peptide-based subunit vaccines. Some of the most promising adjuvants in this regard are combined small molecule Toll-like receptor-7/8 agonists (TLR-7/8a). However, poor pharmacokinetic properties have precluded TLR-7/8a for use in vaccines. In this thesis, polymer carriers were used to control pharmacokinetics and to modulate activity of TLR-7/8a for use as vaccine adjuvants. Combinatorial synthesis and in vivo structure-activity studies were used to evaluate how properties of Polymer-TLR-7/8a conjugates (Poly-7/8a) influence innate immune activation in lymph nodes that drain the site of vaccine administration. The most striking finding was that particle formation by Poly-7/8a strongly enhances the magnitude and duration (>14 days) of innate immune activation in lymph nodes by restricting agonist biodistribution and promoting uptake by dendritic cells. Particle-forming Poly-7/8a optimized for activity were found to induce only local innate immune activation (not systemic) and were effective for eliciting Th1-type CD4 and CD8 T cells that mediated protection against infectious challenge. Based on the importance of particle formation for activity of Poly-7/8a, thermo-responsive Poly-7/8a were developed that exist as single water-soluble macromolecules in solution but undergo temperature-driven particle formation in vivo. In conclusion, polymer carriers of TLR-7/8a represent a versatile and effective platform for modulating innate immune activity and warrant further investigation as a class of adjuvants for vaccines.

For many years the CD45 transmembrane tyrosine phosphatase has been known to be an important molecule in both B and T lymphocyte receptor signalling events. To understand the role of this molecule in lymphocyte ontogeny and function I have created mice which are homozygous for a targeted mutation of exon 12 of the Cd45 gene. These mice have B cells that are unusual in that they appear immature on the basis of their expression of IgM and IgD receptors and yet have increased expression of other markers that are associated with increasing B cell maturation. In addition, CD45-deficient B cells have increased expression of B7.2, suggesting that these cells have been activated. Following BCR stimulation in vitro CD45-deficient B cells have a diminished proliferation response. B-1 cell populations have been found to be greatly reduced in these mice suggesting that CD45 is also a positive regulator of B-1 receptor signalling. The use of the 3-83 transgenic BCR demonstrates that 383/Cd45-/- B cells can be produced however these cells express altered levels of IgM and IgD. Cd45-/- B cells are deleted normally in the presence of the negatively selecting ligand for the transgenic receptor and undergo both Ig heavy and light chain rearrangements normally suggesting that CD45 is not required for these processes. Finally Cd45-/- B cells have a diminished but not absent Ca2+ flux on receptor stimulation that could explain the abolition of some receptor mediated responses. Cd45-/- mice have a severe deficiency in T cell development. Firstly there is a developmental block at the DN3 stage of thymocyte development, suggesting that CD45 plays a role in signalling from the pre-TCR. A second developmental block is at the DP stage in thymocyte developmental which is associated with the positive and negative selection events of T cell selection. Some Cd45-/- T cells are able to migrate to the periphery albeit in greatly reduced numbers. These cells are unusual in that they have an activated phenotype (CD44hi/L-selectinlo). To determine the role of CD45 during positive selection of thymocytes both class 1 and class II transgenic TCR receptors were used. Cd45-/- mice were found to display a profound block in the development of mature T cells expressing the transgenic TCRs suggesting an important role for CD45 in the positive selection of these receptors. Moreover, this deficiency appears to be due to altered thresholds of TCR signalling as assessed using bi-specific antibodies to induce positive selection events in a dose dependent manner. The use of a transgenic TCR to investigate T cell negative selection events demonstrates that Cd45-/- thymocytes can undergo negative selection events induced by the cognate ligand. However, negative selection induced by endogenous (Mtv) and exogenous (SEB) superantigens are deficient in Cd45-/- thymocytes. In addition Cd45-/- T cells are found to have a profound block in their ability to mobilise Ca2+ in response to antigen receptor stimulation.

Despite the chronic debate in the etiology of crohn's disease (cd), a debilitating inflammatory bowel disease (ibd) closely related to ulcerative colitis (uc), an emerging interest in a possible mycobacterial role has been marked. Granuloma and pathologic manifestations in cd resemble aspects found in tuberculosis, leprosy and paratuberculosis. The latter, a chronic enteritis in cattle, goat, sheep and primates, which is similar to human enteritis, also known as cd, is caused by a fastidious, slow growing mycobacterium avium subspecies paratuberculosis (map). Due to the similarities between cd and paratuberculosis, a mycobacterial cause in cd has been proposed. Recent discovery of a possible association between nod2/card15 mutations and risk of cd added support to microorganism-host interactions. In this study, a possible mycobacterial role in cd etiology has been evaluated by investigating the presence of map dna, the state of the cellular immune response and microarray gene expression profiling in peripheral blood and surgical tissue from cd, uc and healthy control subjects. Nested pcr detected map dna in tissue from 10/12(83%) cd patients compared to 1/6(17%) non-ibd subjects. Fluorescence in situ hybridization (fish) with the aid of confocal scanning laser microscopy (cslm) detected map dna in 8/12(67%) cd subjects compared to 0/6(0%) in non-ibd subjects. The detection of map dna by either technique in tissue from cd subjects is significant compared to non-ibd subjects (p < 0.05). Map dna was also detected in both inflamed and non-inflamed tissue from patients with cd suggesting map infiltration in human tissue. Correlation of possible map presence and the function of polymorphonuclear leukocytes (pmn) and peripheral blood mononuclear cells (pbmc) in 19 cd patients and 12 controls have been evaluated. Pmn phagocytosis of viable fitc-map was suppressed in 13/19(68%) cd patients compared to 0/12(0%) in healthy controls (p