Effective control of chronic viral infections may require the generation of polyfunctional T cells (PFCs) which are capable of producing multiple cytokines and possess cytotoxic function. In this study, I investigate (i) whether PFCs play an important role in the long term immune control of a persistent human virus, Epstein-Barr virus (EBV), (ii) the relationship between the development of immunodominance and functionality during the evolution of the anti-viral T cell responses, and (iii) whether PFCs can be generated in patients with EBV-associated haemophagocytic lymphohistiocytosis (HLH). To tackle the first question, I established a 9-color flow cytometry assay to characterize the co-expression of four cytokines (interferon-f [IFN-[], macrophage inflammatory protein 1-] [MIP1-[], tumour necrosis factor-] [TNF- ] and interleukin-2 [IL-2]) and degranulation marker (CD107a) by both EBV lytic and latent peptide-specific CD4+ and CD8+ T cells in 20 healthy long term viral carriers. Two patients with EBV-associated post-transplant lymphoproliferative disorder (PTLD) were studied for comparison. Both CD4+ and CD8+ PFCs were readily generated in the long term carriers with the immunodominant EBV proteins apparently stimulating higher proportions of PFCs than the subdominant viral proteins. The PFCs producing greater amount of cytokines per cell than the single functional T cells. In contrast, EBV-specific PFCs were hardly generated in the patients with PTLD. To investigate the relationship between the development of immunodominance and functionality, I performed a longitudinal study of CD4+ and CD8+ T cell responses in 10 children with infectious mononucleosis (IM) and 4 asymptomatic individuals (AS) with primary EBV infection from the time of diagnosis to one year post-infection. Viral peptide-specific T cells were examined for the co-expression of three cytokines (IFN-t, TNF-T and IL-2), perforin and CD107a upon stimulation with overlapping peptide pools of lytic and latent proteins, respectively. PBMC viral loads were reduced gradually in both IM and AS subjects. Whilst lytic and latent peptide-specific PFCs were still detectable at the acute stage of infection, they showed an increase in functionality over time in response to peptide pools of immunodominant proteins. From acute to persistent infection stage, the CD8+ T cell responses shifted from reactivities against the lytic peptides to those against the latent EBNA3A and 3B peptides with concurrent increase in functionality. Change in CD4+ T cell responses is less obvious, apparently from reactivities towards broad range to EBNA1 peptides. Finally, we found that two patients with the life-threatening EBV-associated haemophagocytic lymphohistiocytosis (HLH) had very high viral loads at the onset of disease. The clinical symptoms improved and viral loads were gradually reduced by the immunosuppressive drug therapy. Interestingly, EBV lytic and latent peptide-specific PFCs could be subsequently generated post-treatment with sustained resolution of clinical symptoms and clearance of plasma viral loads . In conclusion, lytic and latent peptide-specific CD4+ and CD8+ PFCs may confer long term immune control to EBV. The PFCs may be generated concurrent with the establishment of immunodominance hierarchy during the evolution of viral-specific T cell responses. Long term polyfunctional T cell responses to EBV can be formed in patients with EBV-associated HLH. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Glaucoma is a leading cause of world blindness, often associated with elevated eye pressure. Current glaucoma treatments aim to lower eye pressure by improving aqueous humor outflow from the eye. Ocular lymphatics have been demonstrated to contribute to aqueous humor outflow in human and sheep. It is not known whether any glaucoma drugs target this lymphatic drainage. The mouse is a valuable model with similar aqueous humor dynamics and pharmacology as human. Using in vivo hyperspectral fluorescence imaging combined with intracameral quantum dot injection, we identified an ocular lymphatic drainage in mouse. Immunofluorescence and confocal microscopy revealed lymphatic channels in the ciliary body, sclera, and orbit that may be responsible for this lymphatic drainage. We showed that latanoprost, a prostaglandin F2α analog widely used to treat glaucoma, increases this ocular lymphatic drainage. Our findings provide the framework for future development of novel glaucoma drugs that stimulate the ocular lymphatic drainage.

Glaucoma is a leading cause of world blindness, often associated with elevated eye pressure. Current glaucoma treatments aim to lower eye pressure by improving aqueous humor outflow from the eye. Ocular lymphatics have been demonstrated to contribute to aqueous humor outflow in human and sheep. It is not known whether any glaucoma drugs target this lymphatic drainage. The mouse is a valuable model with similar aqueous humor dynamics and pharmacology as human. Using in vivo hyperspectral fluorescence imaging combined with intracameral quantum dot injection, we identified an ocular lymphatic drainage in mouse. Immunofluorescence and confocal microscopy revealed lymphatic channels in the ciliary body, sclera, and orbit that may be responsible for this lymphatic drainage. We showed that latanoprost, a prostaglandin F2α analog widely used to treat glaucoma, increases this ocular lymphatic drainage. Our findings provide the framework for future development of novel glaucoma drugs that stimulate the ocular lymphatic drainage.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available online.

Thesis--University of California, Berkeley, 1961. / "UC-48 Biology and Medicine" -t.p. "TID-4500 (16th Ed.)" -t.p. Includes bibliographical references (p. 149-161).

The lymphatic vasculature is composed of lymphatic endothelial cells (LECs) that coalesce into a branched hierarchy of small capillaries and larger collecting vessels that regulate interstitial fluids, lipid uptake and immunity. Few studies have focused on pulmonary lymphatic system. To fill these critical knowledge gaps, we interrogated the fetal maturation program of lymphatic endothelium, and we provide evidence that CSF1R-lineage progenitors contribute to LECs in the lung during a temporally defined period in early postnatal life. The pulmonary lymphatic system is required for fluid clearance and air breathing at birth, suggesting a prenatal maturation program. To interrogate this, we developed a cell sorting strategy to enrich pulmonary LECs by their unique cell surface immunophenotype (CD45-, EPCAM-, CD31+, VEGFR3+, PDPN+, LYVE1+) for transcriptional profiling. These experiments highlighted the coordinate down-regulation of genes involved in “cell cycle”, and “mRNA processing” along with coordinate upregulation of “complement/coagulation cascade”, “lipid metabolism”, and “angiogenesis” genes from embryonic day E16.5 to E18.5. The most significantly enriched gene set corresponded to the “interferon-alpha/beta signaling” pathway which was confirmed with qRT-PCR and in-situ hybridization. These data provide the first description of the transcriptional landscape of fetal pulmonary LEC maturation. During development, all LECs are thought to originate from embryonic veins, however multiple studies have suggested a myeloid origin for a subset of LECs. A relationship between myeloid cells and the pulmonary LECs has not been elucidated. Here, we used myeloid-specific inducible CSF1R-CreERtdTomato lineage tracing mice and identified rare, single cells that co-expressed CSF1R- CreERtdTomato and Prox1, the master lymphatic regulator, in the postnatal day 3 lung. This process was temporally restricted to the early postnatal period. Lineage tracing with additional myeloid-Cre mice (CSF1R-iCre and CX3CR1-Cre) also showed contribution to postnatal LECs. To determine the biological significance of CSF1R-derived LECs to postnatal lung biology, we performed conditional Prox1 loss of function experiments. CSF1R-CreER mediated deletion of Prox1 resulted in lymphatic hypoplasia, edematous foci and clotting. These findings suggest that early postnatal CSF1R+ progenitors contribute to the pulmonary lymphatic endothelium and that vascular clotting may result from lymphatic malformation/dysfunction. / 2021-06-14T00:00:00Z

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The lymphatic system plays essential roles in regulating fluid balance and immunosurveillance. Across the body, local lymphatic vessels collect waste in the form of lymph and deliver it to nearby lymph nodes (LNs) to be filtered and screened for pathogens. With broad implications in adaptive immunity, cancer metastasis, and cancer treatment, developing novel in vitro models will provide new platforms to explore lymphatic function in health and disease. This dissertation sought to develop tissue-specific engineered models of the LN stroma and the meningeal lymphatics to examine the transport of cells and fluid. Within the LN, fibroblastic reticular cells (FRCs) maintain a network of extracellular matrix conduits that guide varying rates of interstitial fluid flow (IFF) based on inflammatory state. Eventually, that flow exits the LN through the afferent lymphatics, consisting of lymphatic endothelial cells (LECs). We first developed a spatially organized model of the LN stroma consisting of a monolayer of LECs on the underside of a tissue culture insert and an FRC-laden hydrogel within. We demonstrate that high magnitude IFF (3.0 µm/s) had positive impacts on FRCs but disrupted the integrity of the LEC barrier, and these effects were accompanied by increased secretion of a variety of inflammatory chemokines. We also show that IFF of any magnitude decreased T cell egress from the model. Next, we sought to apply the LN stroma model toward understanding metastasis. LN metastasis is the most important prognostic factor in breast cancer, with size of metastasis informing treatment plan. Metastasis greatly alters the structure of the LN, which in turn alters transport. However, the impact of altered transport on cancer progression is not well understood. We added different numbers of breast cancer cells to our LN stroma model to examine tumor burden. We found that tumor cells invaded the LEC barrier at similar numbers regardless of initial burden. Additionally, at the highest tumor burden, diffusivity in the stroma was significantly decreased. Most excitingly, flow velocity was positively correlated with FRC spread in the hydrogel, demonstrating the contributions of FRCs to transport. Finally, we looked to the central nervous system (CNS). The meningeal lymphatics are responsible for draining cerebrospinal fluid to the cervical lymph nodes for CNS immunosurveillance. We developed a simple model of a meningeal lymphatic vessel lumen consisting of a monolayer of LECs on the underside of a tissue culture insert and a monolayer of meningeal fibroblasts within. This is, to our knowledge, the very first in vitro model of the meningeal lymphatics. We demonstrate that our model has barrier function and is capable of immune cell transmigration and egress. We examined how systemic chemotherapy for breast cancer could cause off-target disruption of the meningeal lymphatics and found that docetaxel was significantly deleterious. We further began to explore leukemia cell behavior in our LN stroma and meningeal lymphatics model. Throughout this dissertation, we emphasize the importance of incorporating fluid and cell transport into engineered models of immunity. These models represent a step toward building up the complexity of in vitro lymphatic models to improve pre-clinical screening and understand pathophysiology. / Doctor of Philosophy / The lymphatic system plays essential roles in regulating fluid balance and immune system surveillance. Across the body, local lymphatic vessels collect waste in the form of lymph and deliver it to nearby lymph nodes (LNs) to be filtered and screened for pathogens like viruses or bacteria. With broad implications in immunity, cancer metastasis, and cancer treatment, developing novel models in the lab using human cells and 3-dimensional biomaterials will provide new platforms to explore lymphatic function in health and disease. This dissertation sought to develop engineered models that were specific to the lymph node stroma and the meningeal lymphatics to examine the transport of cells and fluid. Within the LN, fibroblastic reticular cells (FRCs) maintain a network of channels that guide varying rates of interstitial fluid flow (IFF) based on how inflamed the LN is. Eventually, that flow exits the LN through the afferent lymphatics, consisting of lymphatic endothelial cells (LECs). We first developed a spatially organized model of the LN stroma consisting of LECs on the underside of a porous membrane and an FRC-laden hydrogel above the membrane and demonstrated that high magnitude IFF altered morphology, immune cell behavior, and inflammatory protein secretion in the model. Next, we sought to apply the LN stroma model toward understanding cancer metastasis. LN metastasis is the most important prognostic factor in breast cancer, with size of metastasis informing treatment plan. Metastasis greatly alters the structure of the LN, which in turn alters the transport of lymph and immune cells. However, the impact of altered transport on cancer progression is not well understood. We added different numbers of breast cancer cells to our LN stroma model to examine tumor burden and found that tumor cells invaded the LECs at similar rates regardless of initial density, but that diffusion, a transport parameter, was significantly changed by high tumor cell density. Finally, we looked to the central nervous system (CNS). The meningeal lymphatics are responsible for draining cerebrospinal fluid to the cervical lymph nodes to screen for pathogens in the CNS. We developed a simple model of a meningeal lymphatic vessel lumen consisting of LECs and meningeal fibroblasts on either side of a porous membrane. This is, to our knowledge, the very first in vitro model of the meningeal lymphatics. We examined how systemic chemotherapy for breast cancer could cause off-target disruption of the meningeal lymphatics and found that docetaxel was significantly damaging to the model. Throughout this dissertation, we emphasize the importance of incorporating fluid and cell transport into engineered models of lymphatics. These models represent a step toward building up complexity to improve the toolset for pre-clinical screening and studying disease progression.

Although the major functions of the lymphatic system are fairly well defined, its vasculature has yet to be well characterized in comparison to its blood vasculature counterpart. Recent advances in optical imaging techniques have allowed for more detailed and quantitative evaluations of lymph flow dynamics and mechanism. A rat tail is often used for investigations of lymph flow because of the simple geometry, superficial nature, and disease progression models of its collecting lymphatic vessels. In this study, a pressure cuff system was fabricated and coupled with an existing functional near infrared (NIR) imaging system to measure the overall pumping pressure of the lymphatic vessels of a rat tail. In addition to adapting the system for use on rodents, previous systems used for measuring lymphatic pumping pressure in humans were improved upon in several ways. The system defined here utilizes closed-loop feedback control of pressure application at smaller, more precise intervals. Using this device, a significant difference in lymphatic vessel pumping pressure was detected between a control case and a treatment case in which a vasoactive substance with a nitric oxide donor (GTNO ointment) was applied to the tail. Although it is known that nitric oxide plays a crucial physiologic role in propagation of flow through lymphatic vessels, this study has quantified its significant pharmacological reduction of pumping pressure for the first time.