During a T-dependent immune response, activated B cells enter structures called germinal centers (GC) in the follicles of secondary lymphoid organs. GC B cells proliferate and undergo diversification of their Ig through somatic hypermutation and class-switch recombination. These Ig diversifications require the activity of the enzyme activation-induced deaminase (AID). Clonal selection within GCs selects GC B cells with the highest affinities for antigen to mature into plasma cells and memory B cells. GCs are underpinned by stromal cells called follicular dendritic cells (FDC). FDC functions include secretion of B cell chemokines and the retention of Ag complexes that allow GC B cells to test the affinity of their Ig. FDCs require constitutive signaling through lymphotoxin beta receptor (LTβR) from lymphotoxin alpha-beta (LTαβ) on the surface of B cells to maintain their functions. In these studies, I investigated the properties of GCs using two primary experimental models. First, I employed genetic and pharmacological ablation of LTβR signaling to investigate the expression of AID and the function of GCs in the absence of FDCs. I determined that FDCs are not required for GC formation or the expression of AID in lymph nodes, but that FDCs are crucial for affinity maturation. Second, I examined the competition between AID-/- and WT B cells in the GCs of mixed BM chimeras to investigate the role of affinity maturation during clonal selection. I found that AID increases GC B cell apoptosis, likely by inducing DNA damage, and that this is important in regulating GC size. I also found that AID-/- B cells accumulate at the centrocyte stage of the GC reaction and that this is due to a partial block in plasma cell maturation.

The ability of cancer cells to escape anti-tumor immune responses is acknowledged as one of the hallmarks of cancer. Overexpression of immunoregulatory molecules is one mechanism responsible for the immunsuppressive network that is characteristic of the tumor microenvironment. In this thesis, we investigated the role of CD200, a potent immunoregulatory molecule, in Chronic Lymphocytic Leukemia. We showed that functional blockade of CD200 on lymphoma cells or primary CLL cells, both of which express CD200 at high levels, augmented cytotoxic killing of these cells by effector CD8+ T cells in vitro. We also identified and characterized a previously unrecognized soluble form of CD200, sCD200, present in elevated levels in CLL plasma when compared to plasma from controls. The data reported show that patients with high sCD200 levels have more aggressive disease, inferring that sCD200 may be a novel prognostic marker for CLL. The in vivo function of sCD200 was investigated for its ability to support engraftment of CLL splenocytes in NOD.SCID mice. Infusion of sCD200hi CLL plasma, but not sCD200lo normal plasma, enhanced engraftment of CLL-splenocytes in vivo, an effect which was abrogated by depletion of sCD200 from CLL plasma. The prolonged engraftment of CLL cells seen in this model (>6 months) suggests these mice represent a useful pre-clinical model for drug screening. The effect of CD200 blockade was tested in this model, and was found to be as effective in eliminating engrafted CLL cells as rituximab. Investigation of the mechanisms leading to the release of sCD200 from CLL cells showed that sCD200 was produced following ectodomain shedding by ADAM proteases and MMPs. Results from studies reported in this thesis support the hypothesis that CD200 plays a major role in CLL biology, and suggests it may represent a novel therapeutic target for CLL.

The NF kappa B family of heterodimeric transcription factors is activated by many stimuli, and lead to the upregulation of countless genes. Not surprisingly, NF kappa B plays a critical role in many aspects of cellular function. In T and B lymphocytes, antigen receptor stimulation leads to the activation of NF kappa B through a signal transduction cascade involving the Bcl10-MALT1 complex. We hypothesized that this complex may be critical to signalling cascades other than those emanating from antigen receptors. B cell activation factor of the TNF family (BAFF) activates non-canonical NF kappa B heterodimers that promote B cell survival. Here, we show that MALT1 is required for BAFF-induced phosphorylation of NF kappa B2 (p100), p100 degradation and RelB nuclear translocation in B220+ B cells. TRAF3, a known negative regulator of BAFF-R mediated signaling, interacts with MALT1 in a manner which is negatively regulated by BAFF, and TRAF3 levels are enhanced in MALT1-/- B cells. MALT1-/- CD21highCD23low (MZ) B cells show a defect in BAFF-induced survival and MALT1-/- x BAFF-transgenic (Tg) mice have decreased MZ and B1 B cell levels compared to BAFF-Tg mice. In agreement with this in vitro data, phenotypes associated with over-expression of BAFF including increased serum immunoglobulin titres, spontaneous germinal center (GC) formation, and immune complex deposition in the kidney were found to be dependent on B cell-intrinsic MALT1 expression. Our results demonstrate a novel role for MALT1 in biological outcomes induced by BAFF-mediated signal transduction. The mechanism by which the Bcl10-MALT1 complex regulates antigen induced NF kappa B activation in T cells remains controversial. To shed light on this regulatory network, we conducted biochemical purification of Bcl10, and identified Uev1a, a known regulator of antigen receptor mediated NF kappa B activation. We hypothesized that mms2, and structurally similar molecule to Uev1a, may also impinge on NF kappa B activation. Mms2 overexpression in 293T cells inhibited the Bcl10-induced activation of an NF kappa B sensitive luciferase. Lymphocyte development and antigen receptor induced activation occurs normally mms2-/- mice. However, class switched serum immunoglobulins, and survival responses to DNA damage inducing gamma-irradiation, are decreased in mms2-/- mice. Therefore, mms2 is dispensible in vivo for lymphocyte function and development, but is required for DNA damage responses.