<p>Monocytes and monocyte-derived cells are important in providing innate immunity against various pathogens. Monocytes become macrophages or dendritic cells after they enter tissues during inflammation. Macrophages phagocytose microbes and kill them intracellularly in lysosomes. After macrophages are activated, they secret a variety of cytokines as part of innate defense. However, such cytokines have been implicated in causing autoimmune diseases and influenza-induced pathology. For these reasons, we have investigated the role of monocytes and monocyte-derived cells in inducing immune pathology. Moreover, monocytes are also thought to affect adaptive immunity by shaping T cell responses. Yet the enterity of their contributions to adaptive immune response remains to be determined. </p><p>CCR2 is the chemokine receptor required for inflammatory monocytes to enter tissues, and its deficiency in mice has been shown to be protective for influenza-induced immune pathology. We hypothesized that cells that depend on CCR2 to migrate into inflammaed lungs are the cells that induce immune pathology during influenza infection. First, we identified cell types that are recruited to the lungs by CCL2. Similar myeloid cell types, monocytes, monocyte-derived DCs (moDCs), and exudated macrophages (exMAC), also accumulate in the lungs during influenza infection. We then show that these myeloid cells types are derived from monocytes, and that they produce high levels of TNF-&#945; and NOS2. Finally, we show a strong correlation between reduced accumulation of myeloid cells and decreased influenza-induced pathology and mortality in CCR2-deficient mice, suggesting that CCR2 inhibition may be a viable therapy for highly pathogenic influenza infection.</p><p>In the second part of this work, we focus on monocyte-derived dendritic cells in lymph nodes (LN). Inflammatory DCs in LN can arise from moDCs recruited via lymphatics (peripheral moDCs) and from inflammatory monocytes that enter LN directly from the blood (blood-derived moDCs). We examine the role of blood-derived moDCs in inducing LN T cell activation and polarization after immunogenic stimuli. We find that, following viral infection or immunization, inflammatory monocytes are recruited into LN directly from the blood to become CD11c<super>+<super>CD11b<super>hi<super>Gr-1<super>+<super> inflammatory DCs, which produce high levels of IL-12 (p70) and potently stimulate Th1 responses. This monocyte extravasation requires CCR2 but not CCL2 or CCR7. Thus, inflammatory DCs accumulation and Th1 responses are markedly reduced in CCR2<super>-/-<super> mice, preserved in CCL2<super>-/-<super> mice, and relatively increased in CCL19/21-Ser-deficient <italic>plt<italic> mice, in which all other LN DC types are reduced. </p><p>Our findings provide important insights into mutiple roles that monocytes play in both innate and adaptive immunity. Monocytes provide an early response against pathogens. As we now demonstrate, this response can be excessive, leading to a significant immune pathology during influenza infection that has been previously attributed to neutrophils. We also provide the first demonstration that monocytes play an important role in regulating adaptive immune responses. We find that monocyte-derived DCs are both sufficient and necessary for the development of Th1-polarized immune responses within LNs. Taken together, our results demonstrate that the roles played by monocytes in innate immunity adaptive immunity, and immune pathology are much greater than previously appreciated and that regulating monocyte function may be an effective means to regulate certain types of immune responses.</p> / Dissertation

<p>The immune system is constantly faced with the daunting task of protecting the host from a large number of ever-evolving pathogens. In vertebrates, the immune response results from the interplay of two cellular systems: the innate immunity and the adaptive immunity. In the past decades, dendritic cells have emerged as major players in the modulation of the immune response, being one of the primary links between these two branches of the immune system.</p><p>Dendritic cells are pathogen-sensing cells that alert the rest of the immune system of the presence of infection. The signals sent by dendritic cells result in the recruitment of the appropriate cell types and molecules required for effectively clearing the infection. A question of utmost importance in our understanding of the immune response and our ability to manipulate it in the development of vaccines and therapies is: "How do dendritic cells translate the various cues they perceive from the environment into different signals that specifically activate the appropriate parts of the immune system that result in an immune response streamlined to clear the given pathogen?"</p><p>Here we have developed computational and statistical methods aimed to address specific aspects of this question. In particular, understanding how dendritic cells ultimately modulate the immune response requires an understanding of the subtleties of their maturation process in response to different environmental signals. Hence, the first part of this dissertation focuses on elucidating the changes in the transcriptional</p><p>program of dendritic cells in response to the detection of two common pathogen- associated molecules, LPS and CpG. We have developed a method based on Langevin and Dirichlet processes to model and cluster gene expression temporal data, and have used it to identify, on a large scale, genes that present unique and common transcriptional behaviors in response to these two stimuli. Additionally, we have also investigated a different, but related, aspect of dendritic cell modulation of the adaptive immune response. In the second part of this dissertation, we present a method to predict peptides that will bind to MHC molecules, a requirement for the activation of pathogen-specific T cells. Together, these studies contribute to the elucidation of important aspects of dendritic cell biology.</p> / Dissertation

<p>Pin1 is a ubiquitously expressed phosphorylation-specific prolyl isomerase that regulates substrate function by catalyzing the cis-trans isomerization of prolyl bonds. Through this modulation, Pin1 has been shown to influence the stability, localization, and/or activity of a diverse set of protein substrates that participate in a variety of cellular responses, such as cell cycle progression, modulation of cell stress, and apoptosis. In addition to extensive studies in non-hematopoietic cells, Pin1 has also been shown to regulate immune cell function. Indeed, Pin1 participates in germinal center B cell development and eosinophil granulocyte survival. It also facilitates cytokine production in T cells, eosinophil granulocytes, and plasmacytoid dendritic cells. Through specific activities such as these, Pin1 has been demonstrated to modulate responses to viral challenge, respiratory allergens, and organ transplantation. </p><p>Due to previously described functions of Pin1 in regulating cells of both the innate and adaptive immune system, we predicted that Pin1 would participate in systemic inflammatory responses. Upon inducing systemic inflammation in mice, we observed a profound reduction in circulating cytokine concentrations in Pin1-null mice compared to WT mice. This result prompted further investigations, which are described in chapter 3 and chapter 4 of this dissertation. In chapter 3, we evaluate the potential contribution of macrophages to the defects we observe in LPS-challenged Pin1-null mice. Using primary macrophages, bone marrow-derived macrophages, and MEF, we ultimately exclude a role for Pin1 in modulating LPS-induced production of pro-inflammatory cytokines in these cells. In chapter 4, we uncover a defect in the accumulation of conventional dendritic cells (cDC) in LPS-challenged Pin1-null mice. Upon more careful examination of spleen cDC subsets in Pin1-null mice, we discovered a defect in the CD8+ subset. Experiments described in this chapter collectively indicate a role for Pin1 in preferentially modulating late stages of development of the CD8+ subset of cDC. Consistent with such a defect, the expansion of adoptively transferred WT CD8+ T cells was less robust in Pin1-null mice than WT mice upon infection with the bacterium Listeria monocytogenes . At the end of chapter 4, we provide evidence that Pin1 facilitates the degradation of the hematopoietic transcription factor PU.1, and propose that deregulation of PU.1 expression may be one mechanism by which Pin1 modulates CD8+ cDC development. The work described in this dissertation began by evaluating a potential role for Pin1 in modulating pro-inflammatory cytokine production in macrophages; ultimately, however, we uncovered a novel role for Pin1 in preferentially modulating the development of the CD8+ subset of cDC. The results presented herein expand the current understanding of DC development and further implicate Pin1 as an important modulator of both innate and adaptive immune responses.</p> / Dissertation

Over the past several decades, our understanding of the immune system has advanced considerably. With it, an appreciation for its role in a number of diseases, such as cancer and infection has significantly grown. While our increased understanding of the immunological mechanisms underlying these diseases has improved treatment, considerable morbidity and mortality from these illnesses still exists signifying the need for more effective and innovative therapies. Dendritic cell (DC) therapy has been shown to be a promising approach to induce strong immune responses for immunotherapy, and biomaterial-based strategies have been developed to target DCs in vivo to facilitate this purpose. Given the importance of calcium in DC function and activation, we hypothesized that we could develop a biomaterial-based approach to locally and specifically control calcium signaling in DCs in vivo as a novel strategy for immunotherapy. Our first sub-hypothesis was that the calcium used to crosslink alginate gels, a commonly used biomaterial, could activate DCs in vitro; our second sub-hypothesis was that calcium ionophore A23187 could be delivered from biomaterials to activate DCs in vitro; and our third sub-hypothesis was that calcium used to crosslink alginate gels and/or controlled delivery of A23187 could increase local inflammation in vivo. We found that both the calcium released from calcium alginate gels and A23187 matured DCs and enhanced TLR-induced inflammatory cytokine secretion in vitro. Although we were unable to effectively deliver A23187 in vivo, calcium alginate gels injected subcutaneously were able to upregulate a number of inflammatory cytokines and chemokines relative to barium alginate gels. Likewise, when LPS was delivered from calcium alginate gels, the inflammatory effects of LPS on surrounding tissue were enhanced compared to when it was delivered from barium alginate gels. Thus, we confirmed that the calcium crosslinker in alginate gels could activate DCs, and provided a proof-of-principle that calcium signaling could be harnessed in vivo to enhance the immune response. Not only does this work impact the future of biomaterial design, but it may also enhance our understanding of DC biology. This thesis lays the groundwork for a novel and potentially effective strategy for enhancing DC activation in vivo, and suggests that ion signaling pathways in other cell types (both immune and non-immune) could also be targeted using biomaterials. / Engineering and Applied Sciences

Immunotherapy has been proposed as an alternative to conventional cancer therapies due to its reduced toxicity and ability to induce long-lasting anti-tumor immune responses. Dendritic cell (DC) vaccination is one immune-based anti-cancer strategy that has received attention due to the ability of DC to process and present antigen to T lymphocytes to initiate immune responses. However, the clinical efficacy of DC-based immunotherapy against established cancers in humans has been extremely low and despite recent advances, objective response rates in DC vaccine trials are rarely above 10%. This lack of efficacy is due in part to immunosuppressive factors, such as transforming growth factor &beta (TGF-&beta), present in the tumor microenvironment that promote tumor immune escape. Therefore, TGF-&beta represents a major barrier to effective cancer immunotherapy and strategies to neutralize this cytokine may lead to more efficacious DC vaccines.In this study, we employed two small molecule transforming growth factor &beta receptor type I (T&betaRI/ALK5) kinase inhibitors (HTS466284 and SM16) in combination with DC vaccines to treat established TGF-&beta-secreting 4T1 mammary tumors. The results demonstrate that while both inhibitors blocked the effects of TGF-&beta in vitro, HTS466284 by itself or in combination with DC vaccination was unable to consistently control the growth and metastasis of established 4T1 tumors. In contrast, SM16 inhibited the growth of established tumors when delivered orally and suppressed the formation of pulmonary metastases when delivered orally or via daily intraperitoneal (i.p.) injection. The efficacy of SM16 was dependent on cellular immunity as this drug had no effect in immunodeficient SCID mice. Furthermore, orally delivered SM16 in combination with DC vaccination led to complete tumor regression in several mice that correlated with increased T cell infiltration of the primary tumor and enhanced in vitro IFN-gamma production and tumor-specific cytolytic activity by splenocytes. Finally, a suboptimal dose of SM16 that failed to control primary tumor growth on its own synergized with DC vaccination to inhibit the growth of established 4T1 tumors. These findings suggest that blockade of TGF-&beta signaling using a small molecule T&betaRI/ALK5 kinase antagonist may be an effective strategy to bolster the efficacy of DC-based cancer vaccines.

Dendritic cells (DC) are potent antigen presenting cells involved in the initiation of the alloimmune response and organ transplant rejection. This thesis, has investigated pharmacological and genetic approaches to manipulate DC in order to generate tolerogenic DC which elicit poor allostimulatory activity as potential cell therapy agents to treat allograft rejection. In the first aspect of this study, human monocyte-derived DC were used to study the influence of Rapamycin (RAPA) on DC phenotype and function. This study showed that RAPA when added to monocytes prior to DC differentiation or after DC maturation generated tolerogenic DC as evidenced by the ability of these cells to induce T cell hyporesponsiveness. However, T cell hyporesponsiveness was associated with downregulation of costimulatory molecules only when added prior to differentiation and surprisingly was not influenced by the induction of CD4 ⁺FoxP3 ⁺ T cells. To assess the effects of RAPA on DC function in the transplant setting an in vivo chimeric model of ovine vascularised skin allograft transplantation was established in immunocompromised NOD/SCID mice as a host. This model was established as a preliminary model to acquire in vivo data prior to testing the effect of pharmacologically modified DC in the preclinical ovine model of renal allograft transplantation, also established in the host laboratory. Firstly, comparison of ovine DC obtained from cannulation of the prefemoral lymphatic vessels in sheep demonstrated that RAPA-modified ovine DC acted as poor stimulators of allogeneic ovine T cells similar to human DC treated with RAPA. Secondly, in NOD/SCID mice engrafted with ovine skin, the infusion of allogeneic ovine T cells together with RAPA-modified ovine DC reduced histological rejection in comparison to control DC. In the second aspect of this study, the effects of genetic manipulation of DC were investigated. In order to investigate the effects of genetic modification of DC, two isoforms of the human HLA-G molecule, HLA-G1 (membrane bound) and HLA-G5 (soluble isoform) were used to generate adenoviral vectors. Unexpectedly, both HLA-G isoforms expressed by human DC transfectants were unable to induce allogeneic T cell hyporesponsiveness in the mixed lymphocyte reaction (MLR). Surprisingly, in the MLR the allogeneic T cells acquired HLA-G1, but not HLA-G5, indicating that direct cell contact and membrane transfer from DC to T cells occurred (Trogocytosis). In addition to HLA-G1, costimulatory molecules (CD40, CD80, CD86 and MHC Class II) were also cotransferred from DC to allogeneic T cells. Accordingly, in secondary proliferation assays T cells immunoselected after co-culture with allogeneic untransfected DC (TUT) demonstrated potent antigen presenting activity when used as stimulators of autologous T cells (analogous to the indirect pathway of antigen presentation). In contrast to TUT, immunoselected T cells that acquired HLA-G1 (THLA-G1) upon co-culture with DCtransfectants showed poor stimulatory capacity. Thus the data reported in this thesis supports the proposed novel concept that HLA-G acquired by T cells through genetically modified DC, functions to autoregulate T cells via T-T cell interaction through the HLA-G receptor ILT2 (negative signalling receptor) expressed on T cells. In conclusion, this thesis has firstly provided supportive evidence that the pharmacological modification of human and ovine DC with RAPA has potential therapeutic effects on allograft rejection. Secondly, the genetic modification of DC to induce expression of HLA-G has specifically allowed the transfer of this molecule to T cells by trogocytosis and the inhibition of alloreactive T cell expansion. / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2009

The Mater Medical Research Institute proposes to undertake a Phase I clinical trial using CMRF-56 immunoselected blood dendritic cells (BDC) loaded with control and myeloma-associated tumour peptide antigens for the treatment of multiple myeloma (MM) patients with minimal residual disease. This thesis describes some of the fundamental pre-clinical in vitro experiments undertaken in preparation for this trial so as to maximise the potential of this vaccine to induce myeloma-specific immune responses. These experiments involved determining the parameters for optimal activation of the CMRF-56 immunoselected cell preparation and exploring the potential of novel myeloma peptide antigens to induce anti-myeloma cytotoxic T lymphocyte (CTL) responses. CMRF-56 immunoselected cell preparations, containing predominately myeloid BDC, monocytes and B cells, were prepared from both healthy donors and myeloma patients. Activation of this preparation with granulocyte macrophage colony stimulating factor (GM-CSF) was found to increase co-stimulatory molecule expression by and survival of BDC, improve peptide- and lysate-specific CTL induction, and, in combination with prostaglandin E2 (PGE2), improve chemokine-specific migration of BDC. Following optimisation of in vitro CTL generation protocols, GM-CSF activated CMRF-56 immunoselected cells were examined for their ability to induce myeloma-specific immunity. Using lysate from myeloma cell line U266 as an antigen source, a polyclonal T cell pool was generated within which peptide specific CTL recognising myeloma antigens Muc1, HM1.24/BST2, DKK-1 and CT-7/MAGE-C1 could be identified. Furthermore, GM-CSF activated CMRF-56 immunoselected cells pulsed with HLA-A*201 restricted peptides derived from Muc1, HM1.24/BST2 and CT-7/MAGE-C1 could induce CTL capable of lysing both peptide- and myeloma cell line targets in both healthy donors and myeloma patients. These results provide the first evidence of immunogenic HLA-A*201 restricted epitopes of novel myeloma antigen CT-7/MAGE-C1. The data collected in this study supports the application of GM-CSF activated CMRF-56 immunoselected cells loaded with defined myeloma peptide antigens for the therapeutic vaccination of MM patients with minimal residual disease.

Acute graft versus host disease (aGVHD) affects more than 40% of patients undergoing haematopoietic stem cell transplantation. aGVHD occurs after transplantation of donor haematopoietic cells into hosts incapable of rejecting the donor cells, when donor T cells attack host tissue. Despite extensive efforts, aGVHD remains problematic to prevent and difficult to control. Current therapies to prevent aGVHD induce profound immunosuppression, leaving patients at increased risk of infection and leukaemic relapse. Dendritic cells (DC) are professional antigen presenting cells of haematopoietic origin and are the primary stimulators of the immune system, uniquely being able to activate naïve T cells. A growing body of evidence suggests that DC are responsible for the stimulation of the donor T cells which cause aGVHD. I have used a model of aGVHD which utilizes conditioned severe combined immunodeficient mice transplanted with human peripheral blood mononuclear cells (PBMC). In this model human CD4+ T cells appear to be responsible for an aGVHD-like syndrome which results in death 15-30 days post transplant. I have shown, using in vitro depletion of individual populations, that other subpopulations of human PBMC did not affect the survival of the mice. I have also demonstrated that human DC are required for the induction of aGVHD in the majority of mice. This novel finding validated the use of this model to test the primary hypothesis; that antibody mediated depletion of DC would prevent aGVHD. The murine IgM monoclonal antibody (Mab), CMRF-44 Mab, is specific for an unknown molecule expressed on the surface of activated human DC. Previous work had shown that when mixed lymphocyte reaction stimulator cells were depleted of CMRF-44+ cells, there was a significant reduction in the proliferation of responder cells. Here I tested the efficacy of CMRF-44 as a therapy for the prevention of aGVHD in the model. CMRF-44 Mab did not improve survival of mice treated with human PBMC, despite recent data showing that CMRF-44 expression on DC was predictive of aGVHD in patients. In vitro depletion of CMRF-44+ cells from human PBMC prior to transplantation also did not reduce incidence of aGVHD. An alternate target for the depletion of human DC was CD83 which is also expressed on the surface of activated human DC. I generated a rabbit polyclonal antibody using a human CD83 fusion protein, which was then affinity purified in a multi-step process which yielded only antibody specific for human CD83. Treatment with this antibody greatly improved survival of transplanted mice. Further experiments showed that anti-CD83 treatment did not abrogate human leucocytes including CD8+ memory T cells suggesting that a therapy using an anti-CD83 antibody has the potential to prevent aGVHD without the immunosuppression associated with current anti-aGVHD therapies. The work described here has validated the use of a human mouse chimeric model as an in vivo assay of human DC function and shown that targeting CD83 has the potential to reduce the incidence of clinical aGVHD whilst preserving donor memory T cells.

The Mater Medical Research Institute proposes to undertake a Phase I clinical trial using CMRF-56 immunoselected blood dendritic cells (BDC) loaded with control and myeloma-associated tumour peptide antigens for the treatment of multiple myeloma (MM) patients with minimal residual disease. This thesis describes some of the fundamental pre-clinical in vitro experiments undertaken in preparation for this trial so as to maximise the potential of this vaccine to induce myeloma-specific immune responses. These experiments involved determining the parameters for optimal activation of the CMRF-56 immunoselected cell preparation and exploring the potential of novel myeloma peptide antigens to induce anti-myeloma cytotoxic T lymphocyte (CTL) responses. CMRF-56 immunoselected cell preparations, containing predominately myeloid BDC, monocytes and B cells, were prepared from both healthy donors and myeloma patients. Activation of this preparation with granulocyte macrophage colony stimulating factor (GM-CSF) was found to increase co-stimulatory molecule expression by and survival of BDC, improve peptide- and lysate-specific CTL induction, and, in combination with prostaglandin E2 (PGE2), improve chemokine-specific migration of BDC. Following optimisation of in vitro CTL generation protocols, GM-CSF activated CMRF-56 immunoselected cells were examined for their ability to induce myeloma-specific immunity. Using lysate from myeloma cell line U266 as an antigen source, a polyclonal T cell pool was generated within which peptide specific CTL recognising myeloma antigens Muc1, HM1.24/BST2, DKK-1 and CT-7/MAGE-C1 could be identified. Furthermore, GM-CSF activated CMRF-56 immunoselected cells pulsed with HLA-A*201 restricted peptides derived from Muc1, HM1.24/BST2 and CT-7/MAGE-C1 could induce CTL capable of lysing both peptide- and myeloma cell line targets in both healthy donors and myeloma patients. These results provide the first evidence of immunogenic HLA-A*201 restricted epitopes of novel myeloma antigen CT-7/MAGE-C1. The data collected in this study supports the application of GM-CSF activated CMRF-56 immunoselected cells loaded with defined myeloma peptide antigens for the therapeutic vaccination of MM patients with minimal residual disease.

In this study we examined the effect of the tick saliva from I. ricinus and the effect of recombinant protein IRS-2 from the saliva of I. ricinus on dendritic cells derived from the mice bone marrow. We studied their effect on the production of cytokines by dendritic cells after the stimulation by B. burgdorferi, their effect on the expression of genes, that participate in phagocytosis, and the impact of the tick saliva on phagocytosis of B. burgdorferi by dendritic cells.