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Fas Ligand (FasL) binds to the Fas receptor to induce apoptosis or activate other signaling pathways. FasL can also transduce "reverse signals" and thus participate in bidirectional signaling. The FasL intracellular domain contains consensus sequences for phosphorylation and a proline rich protein interaction domain. This latter region of FasL has previously been implicated in FasL reverse signaling and regulation of FasL surface expression. In this report, we sought to identify novel FasL interacting proteins to help understand signaling through and trafficking of this death factor. Using mass spectrometry, we identified sorting nexin 18, adaptin beta, Grb2, PACSIN2 and PACSIN3 as FasL interacting proteins. RNAi mediated knockdown of Grb2 significantly reduced the surface expression of FasL and increased its expression intracellularly. Our data show that Grb2 controls the subcellular localization of FasL. All other proteins identified in our screen could be classified as trafficking-associated proteins, highlighting the complex regulation of the surface expression of this death factor.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 82-102).

Thesis (Ph.D.). / Written for the Dept. of Physiology. Title from title page of PDF (viewed 2008/01/17). Includes bibliographical references.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 71-91).

Thesis (Ph.D. in Immunology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 151-168). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

The instructive signals received by T cells during the programming stages of activation will determine the fate of effector and memory populations generated during an immune response. Members of the tumor necrosis factor (TNF) superfamily play an essential role in influencing numerous aspects of T cell adaptive immune responses including cell activation, differentiation, proliferation, survival, and apoptosis. My thesis dissertation describes the involvement of two such members of the TNF superfamily, TNF and FasL, and their influence on the fate of T cells early during responses to viral infections and to the induction of transplantation tolerance. TNF is a pleiotropic pro-inflammatory cytokine that has an immunoregulatory role in limiting the magnitude of T cell responses during a viral infection. Our laboratory discovered that one hallmark of naïve T cells in secondary lymphoid organs is their unique ability to rapidly produce TNF after activation and prior to acquiring other effector functions. I hypothesized that T cell-derived TNF will limit the magnitude of T cell responses. The co-adoptive transfer of wild type (WT) P14 and TNF-deficient P14 TCR transgenic CD8+ T cells, that recognize the GP33 peptide of lymphocytic choriomeningitis virus (LCMV), into either WT or TNF-deficient hosts demonstrated that the donor TNF-deficient P14 TCR transgenic CD8+ T cells accumulate to higher frequencies after LCMV infection. Moreover, these co-adoptive transfer experiments suggested that the effect of T cell-derived TNF is localized in the microenvironment, since the TNF produced by WT P14 TCR transgenic CD8+ T cells did not prevent the accumulation of TNF-deficient P14 TCR transgenic CD8+ T cells. To determine if T cell-produced TNF is acting on professional APC to suppress the generation of virus-specific T cell responses, I performed co-adoptive transfer experiments with WT P14 TCR transgenic CD8+ and TNF-deficient P14 TCR transgenic CD8+ T cells into TNFR1/2 (1 and 2) deficient mice. These experiments demonstrated that the absence of TNFR1/2 signaling pathway in the host cells resulted in a greater accumulation of WT P14 TCR transgenic CD8+ T cells, thereby considerably diminishing the differences between donor WT P14 TCR transgenic CD8+ and donor TNF-deficient P14 TCR transgenic CD8+ T cells. The increased frequency and absolute numbers of WT P14 TCR transgenic CD8+ T cells in TNFR1/R2 deficient recipients suggests that one mechanism for the suppressive effect of T cell-derived TNF on antigen-specific T cells occurs as a result of TNFR signaling in the host cells. However, the donor TNF-deficient P14 TCR transgenic CD8+T cells still accumulated to higher frequency and numbers compared to their donor WT transgenic counterparts. Together, these findings indicate that T cell-produced TNF can function both in an autocrine and a paracrine fashion to limit the magnitude of anti-viral T cell responses. Given the immunoregulatory role of TNF and the ability of peripheral naïve T cells to produce this cytokine, I questioned at what stage of development do T cells become licensed to produce this cytokine. The peripheral naïve T cell pool is comprised of a heterogeneous population of cells at various stages of development, a process that begins in the thymus and is completed after a post-thymic maturation phase in the periphery. I hypothesized that naïve T cells emigrating from the thymus will be competent to produce TNF only after undergoing a maturation process in the periphery. To test this hypothesis, I compared cytokine profiles of CD4+ and CD8+single positive (SP) thymocytes, recent thymic emigrants (RTEs) and mature-naïve (MN) T cells during TCR activation. SP thymocytes exhibited a poor ability to produce TNF when compared to splenic T cells despite expressing similar TCR levels and possessing comparable activation kinetics with respect to the upregulation of CD25 and CD69 following stimulation. The reduced ability of SP thymocytes to produce TNF correlated with a decreased level of detectable TNF message following stimulation when compared to splenic counterparts. Stimulation of SP thymocytes in the context of a splenic environment did not fully enable TNF production, suggesting an intrinsic defect in their ability to produce TNF as opposed to a defect in antigen presentation. Using a thymocyte adoptive transfer model, I demonstrate that the ability of T cells to produce TNF increases progressively with time in the periphery as a function of their maturation state. RTEs identified by the expression of green fluorescent protein (GFP) (NG-BAC transgenic mice), showed a significantly enhanced ability to express TNF relative to SP thymocytes, but not to the extent of MN T cells. Together, these findings suggest that TNF expression by naïve T cells is regulated via a gradual licensing process that requires functional maturation in peripheral lymphoid organs. This highlights the functional heterogeneity of the naïve T cell pool (with respect to varying degrees of TNF production) during early T cell activation that can contribute to the many subsequent events that shape the course of an immune response. The productive activation of naïve T cells requires at least initial two signals; the first being through the TCR and the second is the engagement of co-stimulatory molecules on the surface of the T cells. T cells activated in the absence of co-stimulation become anergic or undergo cell death. Agents that block co-stimulation of antigen-specific T cells are emerging as an alternative to immunosuppressive drugs to prolong allograft survival in transplant recipients. Targeted blockade of CD154-CD40 interactions using a αCD154 monoclonal antibody (MR1) with a simultaneous transfusion of allogeneic splenocytes (donor specific transfusion or DST) efficiently induces tolerance to allografts. This co-stimulation blockade-induced tolerance is characterized by the deletion of host alloreactive T cells within 24 hours of treatment. Toll-like receptor (TLR) agonists abrogate tolerance induced by co-stimulation blockade by impairing the deletion of host alloreactive T cells and resulting in allograft rejection. The goal of my study was to determine the underlying molecular mechanisms that protect host alloreactive T cells from early deletion after exposure to TLR agonists. I hypothesized that TLR ligands administered during co-stimulation blockade regimen differentially regulate the expression of pro- and anti-apoptotic molecules in alloreactive T cells, during the initial stages of activation thereby preventing deletion. To test this hypothesis, I used syngeneic bone marrow chimeric mice containing a trace population of alloreactive KB5 TCR transgenic CD8+ T cells (KB5 Tg CD8+ T cells) that recognize H-2Kb as an alloantigen. I show here that KB5-CD8+ T cells downregulate CD127 (IL-7R!) and become apoptotic as early as 12 hrs after co-stimulation blockade. In contrast, KB5 Tg CD8+ T cells from mice treated with bacterial lipopolysaccaride (LPS) during co-stimulation blockade failed to become apoptotic, although CD127 was downregulated. Examination of the mRNA expression profiles of several apoptotic genes in purified KB5 CD8+ T cells from mice treated with DST+anti-CD154 for 12 hrs revealed a significant upregulation of FasL mRNA expression compared to the untreated counterparts. However, in vitro FasL blockade or in vivo cytotoxicity experiments with mice deficient in Fas or FasL indicated that the Fas-FasL pathway might not be crucial for tolerance induction. Another pro-apoptotic molecule BIM was upregulated in alloreactive T cells during co-stimulation blockade. This suggests that both the Fas pathway and BIM may be playing complementary roles in inducing deletional tolerance. Although FasL expression was diminished in alloreactive T cells in the presence of LPS, BIM expression was not diminished, suggesting that alloreactive T cells may still be vulnerable to undergo apoptosis. Concomitantly, I also found that LPS treatment during co-stimulation blockade resulted in non-specific upregulation of Fas expression in alloreactive T cells and non-transgenic T cells (CD4+ and CD8+). I demonstrate here that treatment with Fas agonistic antibody in vitrofor 4 hours can selectively induce apoptosis of alloreactive T cells that were believed to be refractory to apoptosis during LPS treatment. I speculate that under these conditions, deletion may be occurring due to the involvement of both Fas and BIM. Further, the mRNA expression profile revealed interleukin-10 (IL-10) as a molecule induced in alloreactive T cells during LPS treatment. Analysis of serum confirmed the systemic expression of IL-10 protein in mice treated with LPS during co-stimulation blockade. I hypothesized that LPS-induced IL-10 can have an anti-apoptotic role in preventing the deletion of alloreactive T cells and mediating allograft rejection. Contrary to my hypothesis, I found that IL-10 KO mice rejected allogeneic target cells similar to their WT counterparts, suggesting that IL-10 may not be required for LPS-mediated abrogation of tolerance induction. In addition to the systemic induction of IL-10, LPS also induced cytokines such as interleukin-6 (IL-6), TNF and interferon-γ (IFN-γ). These findings suggest that both Fas-FasL and BIM mediated apoptotic pathways may play complementary roles in inducing the early deletion of activated alloreactive T cells during tolerance induction. On the other hand, the mechanism of LPS mediated abrogation of tolerance induction can not be attributed to IL-10 alone as it may be playing a synergistic role along with other proinflammatory cytokines that may in turn result in the prevention of alloreactive T cell death during this process. Most importantly, these findings indicate that despite emerging from a pro-inflammatory cytokine milieu, alloreactive T cells are still susceptible to undergo Fas-mediated apoptosis during the first 24 hours after co-stimulation blockade and LPS treatment. Therefore, targeting the Fas-FasL pathway to induce deletion of alloreactive T cells during the peri-transplant period may still be a potential strategy to improve the efficacy of co-stimulation blockade induced transplantation tolerance during an environmental perturbation such as inflammation or infection.

Prior immunity to a related or unrelated pathogen greatly influences the host’s immune response to a subsequent infection and can cause a dramatic difference in disease course, a phenomenon known as heterologous immunity. Heterologous immunity can influence protective immunity, immunopathology and/or immune deviation of cytokine-producing T cell subsets. Examples of heterologous immunity have been well documented in mouse models, as well as during human infections. For example, prior immunity to lymphocytic choriomeningitis virus (LCMV) provides partial protection against vaccinia virus (VV), as LCMV-immune mice show reduced VV titers and increased survival upon lethal dose VV infection. Heterologous protection against VV challenge, as a result of LCMV immunity, is mediated by LCMV-specific CD4 and CD8 T cells, as transfer of LCMV-specific memory T cells can mediate this protective effect in naïve mice. The recognition of a single TCR with more than one MHC-peptide complex is referred to as T cell cross-reactivity. A VV Kb-restricted epitope a11r198 was identified to be able to induce cross-reactive responses from LCMV-specific CD8 T cells. During VV infection, LCMV-specific memory T cells that are cross-reactive to VV epitopes produce IFN-γ early in VV infection. IFN-γ is essential for mediating the protection against VV in LCMV-immune mice, as this heterologous protection is absent in IFN-γR-/-and IFN-γ blocking antibody-treated LCMV-immune mice. In addition to protective immunity, cross-reactive LCMV-specific memory T cells and IFN-γ also induce an altered immunopathology during heterologous VV challenge. LCMV-immune mice show moderate to severe levels of inflammation of the fat tissue, known as panniculitis, in the visceral fat pads upon VV challenge. In humans, panniculitis is a painful condition, most commonly presenting as erythema nodosum. Erythema nodosum is a disease of unknown etiology with no known treatment. It may occur following intracellular bacterial and viral infections, and occasionally happens after vaccination with VV for smallpox. During infections there can be a delicate balance between the ability of immune responses to provide protective immunity, and the tendency to induce immunopathology. By using the mouse model of heterologous immunity between LCMV and VV, we tried to understand how the immunity to LCMV biased the balance between the protective immunity and immunopathology, and what effector molecules were responsible for the pathogenesis of panniculitis in this system. TNF is a pleiotropic cytokine, which is required for normal innate and adaptive immune responses. Its functions range from inducing proliferative responses including cell survival, to destructive responses such as promoting apoptosis and programmed necrosis. In response to inflammatory stimuli, activated macrophages/ monocytes produce large amounts of TNF, and upon activation, T cells, B cells and NK cells also produce TNF. In vitro and in vivo studies have shown that TNF in synergy with IFN-γ plays an important role in mediating host defense against pathogens, such as Listeria monocytogenesand poxviruses in mice and hepatitis B virus and human immunodeficiency virus in humans. However, inappropriate expression of TNF often results in tissue damage. Considering the important role TNF plays in both host defense and mediating autoimmune diseases, we hypothesized that TNF was required for mediating both protective and pathogenic effects in the heterologous immunity between LCMV and VV. We first examined whether TNF was involved in mediating protective heterologous immunity. LCMV-immune mice, that were TNF-deficient as a consequence of genetic deletion (TNF-/-) or receptor blockade by treatment with etanercept (TNFR2: Fc fusion protein), were challenged with VV. These TNF-deficient mice showed normal recruitment and selective expansion of cross-reactive LCMV-specific memory CD8 T cells. They also exhibited efficient clearance of VV similar to LCMV-immune mice with normal TNF function. Thus, we concluded that neither TNF nor lymphotoxin (LT), which uses the same receptors as TNF, was required in mediating protective heterologous immunity against VV. Indeed, prior immunity to LCMV could completely compensate for the role of TNF in protection of naïve mice against VV infection, even under conditions of lethal dose inoculum. Thus, heterologous immunity may help explain why treatment of humans with etanercept is reasonably well tolerated with relatively few infectious complications. One of the histological characteristics of panniculitis is necrosis of adipose tissue. It is known that three members in the TNF superfamily, i.e. TNF/LT, FasL and TRAIL are able to induce necrosis of a target cell. It is also known that TNF is able to induce VV-infected cells to go through necrosis, when apoptosis is blocked in these cells by VV protein. Furthermore, TNF and FasL have already been shown to be associated with some skin and fat pathology. Thus, we hypothesized that TNF, FasL and TRAIL were involved in the pathogenesis of panniculitis in VV infected LCMV-immune mice. By using blocking antibodies or genetically deficient mice, we demonstrated that both TNF/LT and FasL were crucial for inducing panniculitis. Although TNFR1 has been reported to induce programmed necrosis, our data indicated that TNFR2, not TNFR1, was involved in mediating tissue damage in the fat pads of LCMV-immune mice infected with VV. We also found that TNF signaled through TNFR2 to up-regulate the expression of Fas on adipocytes. Thus, the engagement of Fas on the adipocytes with FasL expressed on activated VV-specific and cross-reactive LCMV-specific CD8 T cells in the fat pads could lead to panniculitis. Thus, our data may identify a potential mechanism in the pathogenesis of human panniculitis, and may suggest a possible treatment for this painful disease. Recent reports suggest that heterologous immunity may contribute to the tremendous variation in symptoms between individuals, from subclinical to death, upon viral infection. Even in genetically identical mice, variations in immunopathology from none to life-threatening levels of pathology are observed in LCMV-immune mice during VV infection. By adoptive transfer of splenocytes from a single LCMV-immune donor into two recipients, we showed that similar levels of pathology were generated in mice receiving the same splenocytes. However, the level of pathology varied among recipients receiving splenocytes from different LCMV-immune donors. The difference in levels of VV-induced pathology observed in individual LCMV-immune mice was a reflection of the private specificity of the T cell repertoire, which is a unique characteristic of each individual immune host. The goal of this doctoral thesis is to understand how heterologous immunity contributes to the pathogenesis of panniculitis. Our data demonstrate that TNF/LT and FasL directly contribute to development of panniculitis in LCMV-immune mice during VV infection, and suggest that anti-TNF treatment might be a useful treatment for diseases, such as erythema nodosum and lupus-induced acute fatty necrosis in humans.