Pauline Johnson

 
Prospective Graduate Students / Postdocs

This faculty member is currently not looking for graduate students or Postdoctoral Fellows. Please do not contact the faculty member with any such requests.

Professor

Research Classification

Diseases of the Immune System

Research Interests

Immunology
Inflammation
Immune-cancer interactions
Innate immunity
Macrophages and myeloid cells

Relevant Thesis-Based Degree Programs

Affiliations to Research Centres, Institutes & Clusters

 
 

Research Methodology

Flow cytometry
Disease models
in vivo and in vitro analysis

Graduate Student Supervision

Doctoral Student Supervision

Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.

Metabolic regulation of the anti-helminth CD4+ T cell response (2020)

Cellular metabolism is intricately linked to the activation and effector function of CD4+ T cells. Activation triggers an increase in glycolysis, which is thought to fuel the necessary biosynthetic demands of growth and proliferation. Recent work suggests that glycolytic activity further coordinates distinct functions in different T helper subsets. Thus far, however, little has been elucidated about the regulation of metabolism in Th2 cells. Using in vitro cultures and in vivo infection models, I compared glycolysis in Th1 and Th2 cells generated from cytokine-reporter mice. This revealed that Th2 cells from infected mice maintain a low glycolytic rate that more closely resembles that of naïve T cells. I hypothesized Th2 cells may be metabolically suppressed by helminths during infection, and therefore tested the capability of purified helminth products to alter glycolysis of in vitro polarized Th2 cells. Cytokine production was inhibited, but an effect on glycolysis could not be consistently demonstrated. However, signalling through the metabolic regulator, mTOR, was reduced in T cells from helminth infected mice compared to controls, supporting the possibility of metabolic modulation in vivo. I questioned whether Th2 cell glycolysis was regulated after exiting the lymph node. By assessing Th2 cell metabolism from the effector site, I discovered that fatty acid metabolism, and not glycolysis, was upregulated in the peripheral tissue. Fatty acid uptake was enhanced in cells expressing the IL-33 receptor, ST2. These cells highly co-expressed programmed death protein 1 (PD-1), a known regulator of T cell metabolism. Hence, I predicted that PD-1 signalling promotes ST2 expression in the tissue. Accordingly, ex vivo stimulation of sorted Th2 cells through PD-1 increased ST2 expression. From these data I propose that PD-1 signalling overcomes a metabolic checkpoint to permit alarmin-responsiveness and tissue-localized cytokine production. Overall, these findings illustrate Th2 metabolism is dynamic in vivo, and that fatty acid metabolism is the predominant pathway regulated during effector differentiation. Importantly, these results argue that immunometabolism data from in vitro systems may not be applicable in vivo. Continuing to understand in vivo Th2 cell metabolism could be pertinent to the future design of anti-helminth treatments, which are urgently needed.

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The regulation of erythroid progenitors and T cells by CD45 (2020)

The immune system plays a crucial role in the protection of the host against infection and the maintenance of homeostasis under normal conditions. This is achieved by cooperative work amongst a diverse population of immune cells. The pan-leukocyte marker CD45 is a receptor-type tyrosine phosphatase expressed on all nucleated hematopoietic cells. While the function of CD45 on adaptive immune cells has been well described, the role of CD45 on innate immune cells and erythroid progenitors is less understood.CD71⁺TER119⁺ erythroid progenitors are prevalent in neonates; in addition to generating mature erythrocytes, they create an immunosuppressive environment. Here, I show that CD45 regulates late erythroid development as CD45-deficient mice maintained a high level of CD71⁺TER119⁺ progenitor cells in the spleen into adulthood. Despite the increase, CD45-deficient mice had normal numbers of mature red blood cells (RBCs) due to increased sensitivity of erythroid progenitors to erythropoietin (EPO) and an increased number of EPO-producing red pulp macrophages (RPMs). Increased CD71⁺TER119⁺ cells in CD45-deficient RAGKO mice (CD45RAGKO) had an impact on the outcome of T cell transfer colitis. CD45RAGKO had delayed systemic wasting and reduced TNFα production by splenic myeloid cells despite having an equal level of inflammation in the colon. Adoptive transfer of erythroid progenitors from CD45RAGKO donor into RAGKO attenuated the weight loss and reduced TNFα expression by RPMs. Co-culturing of erythroid cells suppressed TNFα expression from RPMs in a phagocytosis-dependent manner. These findings implicate CD45 as a positive regulator of systemic inflammation and suggest erythroid progenitors are an anti-inflammatory agent in colitis.

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Regulation of the CD4+ T cell response to influenza infection (2019)

Surviving influenza infection requires a balance of pro- and anti- inflammatory signals, to promote viral clearance while preventing immunopathology. CD4+ T cells are central to this balance, exerting positive and negative influences on the immune response to infection. Since dysregulated CD4+ T cell responses are detrimental to survival, understanding the mechanisms controlling CD4+ T cell function during infection is essential. This thesis examines two aspects of CD4+ T cell regulation, the effect of the immunoregulatory cytokine IL-27 on CD4+ T cell function and the epigenetic control of the CD4+ T cell response to influenza. In chapter 3, I show that IL-27 signalling can regulate the immune response to influenza by inducing IL-10 from effector CD4+ T cells in primary and recall infection. Since regulated IL-10 expression is important for a protective Th1 response, I investigated the mechanism through which IL-27 promotes CD4+ T cell derived IL-10. I found that IL-27 signalling enhances IL-10 expression from effector CD4+ T cells in a primary response but memory cells lose their responsiveness to IL-27. However, these memory cells re-express IL-10 in a recall response to influenza due to an IL-27 induced permissive epigenetic signature deposited at the Il10 locus during primary activation. In chapter 4, I investigated the effect of IL-27 on functional specialisation in Tregs during infection. Upon exposure to IL-27, airway Tregs exhibited a Th1 adapted phenotype characterised by increased T-bet, CXCR3 and IL-10 expression. In Chapter 5, I investigated the link between histone modifications and gene-expression in naïve, effector and memory CD4+ T cells. I found that dynamic changes in histone modifications at gene promoters are associated with temporal gene expression patterns and that super-enhancer target genes encode highly expressed lineage specific determinants in naïve and effector cells. I also show that memory cells contain a subset of primed effector genes and re-express key genes specifying naïve cell identity. Collectively, my findings indicate that IL-27 regulates effector CD4+ and Treg cell function in the lung and that the control of gene expression in effector and memory CD4+ T cells can be achieved through epigenetic modifications at promoter and super-enhancer elements.

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Determining the role of hyaluronan as an environmental cue for macrophages and dendritic cells (2018)

CD44 is a cell surface glycoprotein that binds to hyaluronan (HA), an extracellular matrix glycosaminoglycan. Immune cells widely express CD44, but only a few types of cells such as alveolar macrophages (AMФ) in the lung alveoli bind fluorescein-conjugated HA constitutively. During inflammation and immune responses, other immune cells such as monocytes are activated and can gain the ability to bind HA. However, the functional significance of CD44 and HA interactions remains unclear. Therefore, the aim of this study was to investigate the function of CD44 and HA binding and how they regulate immune cells such as AMФ in the tissue environment. HA has been described as a regulator of tissue inflammation, with HA fragments reported to stimulate immune cells. To test if HA fragments can induce inflammation or are consequences of inflammation, I stimulated macrophages and dendritic cells with various sizes of HA from different sources. Pharmaceutical grade HA and endotoxin-free HA fragments failed to stimulate an inflammatory response in vitro and in vivo, demonstrating they were not pro-inflammatory. Since AMФ constitutively bind HA, I then compared these cells from CD44+/+ and CD44-/- mice to study the role of CD44 and HA binding as regulatory environmental cues. Using adoptive transfer experiments and a mouse model of inflammation, I found CD44 expression and HA binding were required for the survival of mature AMФ, but not for the recruitment and differentiation of monocytes into AMФ. CD44 expression by AMФ was required for a cell surface HA coat, which maintained AMФ survival and numbers in the lung. Since AMФ are essential for regulating pulmonary surfactant lipid homeostasis, CD44 deficiency and the partial loss of AMФ in CD44-/- mice disrupted lipid homeostasis in their lungs. CD44-/- mice had elevated lung surfactant phosphatidylcholine levels and CD44-/- AMФ exhibited an abnormal phenotype and accumulated cellular lipid droplets. They also suffered greater inflammation caused by oxidized phosphatidylcholine. Thus, CD44 deficiency led to a reduction of AMФ numbers and caused intrinsic defects in AMФ surfactant lipid homeostasis. Together, these results demonstrate a role of CD44 and HA binding in maintaining AMФ and lung homeostasis.

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Hyaluronan binding and CD44 in regulating hematopoiesis and CD8 T cell response (2017)

CD44 is a ubiquitously expressed transmembrane glycoprotein. Through CD44, hematopoietic cells can be induced to bind hyaluronan, a component of the extracellular matrix, at specific developmental or functional stages, leading to the hypothesis that the interaction between CD44 and hyaluronan is important in regulating development and function of immune cells. I demonstrated that chondroitin sulfation of CD44 differentially regulates hyaluronan binding by classically and alternatively activated bone marrow-derived macrophages. T cells also increase hyaluronan binding upon activation, and chondroitin sulfate is likely involved in this regulation, since inhibitors of glycosylation, actin rearrangement, or sialylation had no effect on hyaluronan binding by activated T cells. By using competitive cell transfer models, I found CD44 expression and hyaluronan binding were involved in regulating CD8 memory T cell formation and hematopoietic reconstitution. CD44-/- OT-I CD8 T cells formed significantly more memory cells than CD44⁺/⁺ OT-I CD8 T cells in the lymphoid organs, despite forming similar effector cell numbers, after intravenous infection with ovalbumin-expressing Listeria monocytogenes. While in competition, hyaluronan-binding CD8 effector T cells had increased pAkt expression and glucose uptake, both of which negatively regulate memory potential. Furthermore, hyaluronan-binding CD8 effector T cells showed increased death. Overall, this work implicates CD44 and hyaluronan binding as negative regulators of survival through contraction and memory formation by high affinity OT-I CD8 T cells.To investigate the role of hyaluronan binding in hematopoiesis, I transduced CD44-/- bone marrow cells with CD44 point mutants with increased or abolished hyaluronan binding (GOF or LOF, respectively), and then transferred these cells into lethally irradiated hosts in competition with wild type cells. GOF cells out-competed wild type cells, which in turn out-competed LOF cells, in the reconstitution of all myeloid and most lymphoid populations, suggesting a competitive advantage in early hematopoiesis. Within the bone marrow stem and progenitor cells, GOF cells out-competed wild type cells, which in turn out-competed LOF cells, suggesting a role of the CD44-hyaluornan interaction in positively regulating reconstitution by hematopoietic stem and progenitor cells. Overall, this study identified novel roles for CD44 and HA binding in regulating CD8 T memory, and hematopoietic stem and progenitor cells.

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Regulation of Intestinal Inflammation by CD45 (2015)

Factors secreted by intestinal immune cells such as retinoic acid and cytokines are crucial in maintaining homeostasis in the gut. Dysregulation in the secretion of these factors can lead to inflammation and development of colitis. CD45 is a leukocyte specific tyrosine phosphatase important for T cell development and antigen receptor signaling. Here, I show that upon DSS-induced colitis, CD45-/- mice unexpectedly have significant numbers of T cells in their colon and exhibit more severe colitis. This was attributed to increased expression of the gut homing molecule, α4β7 and increased production of IFNγ and IL17A by the CD45-/- T cells. However, in the absence of adaptive immunity, CD45 is required for optimal intestinal innate immune responses. CD45-/- innate lymphoid cells had decreased IL-22 and GM-CSF production and CD45-/- myeloid cells have lower retinoic acid production. This led to less severe colitis when CD45 ⁺/⁺ T cells were transferred into CD45RAG-/- mice as it led to reduced expression of gut homing molecules and reduced homing of T cells to the colon. This defect was corrected by the addition of GM-CSF, which restored retinoic acid production. Induction of colitis by the transfer of naïve T cells into RAG-/- and CD45RAG-/- mice delayed the development of systemic disease in CD45RAG-/- mice, but led to comparable intestinal inflammation at the RAG-/- weight loss endpoint and significantly greater inflammation at the CD45RAG-/- endpoint, corresponding with increased CD45-/- myeloid cells in the colon. Since there was no difference in Foxp3+ regulatory T cells systemically, other options of inhibition of systemic inflammation in CD45RAG-/- mice were explored. CD45RAG-/- mice had increased CD71+TER119+ erythroid cells in the spleen prior to and post colitis induction and failed to downregulate erythroid progenitors upon T cell induced colitis. These suppressive erythroid cells may contribute to the delayed systemic inflammation in these mice. Overall, these results show novel roles for CD45 in the regulation of innate and adaptive immune cell cytokine production, as well as in erythrocyte maturation.

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CD44 and Hyaluronan Binding in Macrophages and Dentric Cells (2013)

Hyaluronan is a glycosaminoglycan present in pericellular and extracellular matrices. Hyaluronan production and binding are tightly regulated during an immune response yet its effects on immune cells, particularly macrophages and dendritic cells, are not fully understood. CD44 is the major surface receptor for hyaluronan, but not all CD44-expressing cells have the ability to bind hyaluronan. Here I demonstrate that hyaluronan binding is dynamically regulated during macrophage activation as well as dendritic cell differentiation, and CD44 provides macrophages with a selective advantage in the alveolar space that correlates with hyaluronan binding. Inflammatory macrophages activated with lipopolysaccharide plus interferon gamma bound higher levels hyaluronan compared to alternatively activated macrophages polarized with interleukin 4 by modifying chondroitin sulfation on CD44, and these macrophages differ in their ability to take up hyaluronan. In GM-CSF bone marrow-derived dendritic cell cultures, hyaluronan binding identified an immature population that was CD11c+ MHC IImid/low, highly proliferative and did not mature into MHC IIhigh cells. The removal of hyaluronan with hyaluronidase inhibited proliferation of the hyaluronan binding cells and promoted the maturation of dendritic cells. Furthermore, the hyaluronan binding cells had several macrophage characteristics and were retained in the alveolar space while the MHC IIhigh, low hyaluronan binding dendritic cells migrated to the lymph node upon inflammation. These results suggest a role for hyaluronan in cell proliferation and retention in the alveolar space. In vivo, peritoneal, splenic and lung interstitial macrophages bound low or no detectable levels of hyaluronan whereas alveolar macrophages constitutively bound high levels of hyaluronan. The alveolar space promoted hyaluronan binding, as instillation of peritoneal macrophages into the lung upregulated their ability to bind hyaluronan. During lung inflammation, the hyaluronan binding population in the alveolar space was initially reduced and then restored upon resolution as a result of macrophage proliferation. In competition assays, CD44 provided hyaluronan binding macrophages with an advantage in the alveolar space. Collectively, this study reveals a novel role for CD44 in the maintenance of macrophages in the alveolar space and suggests that hyaluronan binding is induced at GM-CSF rich environments to help retain tissue macrophages at the site and promote their self-renewal.

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Hyaluronan bindingby CD44 on T cells (2010)

CD44 is a transmembrane protein that binds to hyaluronan, a component of the extracellular and pericellular matrices. Hyaluronan supports cell migration and proliferation during embryonic development, wound repair, as well as tumourigenesis. Hyaluronan binding to CD44 can also regulate leukocyte migration and adhesion. On naïve CD4 and CD8 T cells, CD44 is in its inactive form, but it is upregulated and induced to bind hyaluronan upon T cell activation. High CD44 expression is used as a marker for effector and memory T cells, and recent evidence has implicated CD44 in the formation of CD4 but not CD8 memory T cells. However, it is not clear if effector T cells bind hyaluronan and unknown if memory T cells bind hyaluronan. Hyaluronan binding has additionally been shown to mark a subset of the most suppressive regulatory CD4 T cells and here hyaluronan promotes FoxP3 expression, but the significance of why only a subset of these cells binds hyaluronan is unclear. Thus, the current understanding of when and on which cells hyaluronan binding is induced during an immune response, as well as its function on T cells, is incomplete.The first aim of this work was thus to determine when CD4 and CD8 T cells bind hyaluronan. T cells were activated in vitro with PMA and ionomycin and in vivo during an immune response to Listeria monocytogenes. Hyaluronan binding was assessed over a time course and found to occur on the mostly highly proliferative, activated T cells, as well as on a subset of memory T cells. The second aim of this work was to determine the consequences of binding hyaluronan. Hyaluronan binding on activated T cells was found to enhance their adhesion to fibronectin and inhibited both chemokinesis and chemokine-induced migration. Furthermore, hyaluronan induced chemokine-independent polarization of CD44, but inhibited CD44 co-polarization with phosphorylated ERM proteins. Together, the data suggests that hyaluronan binding is induced on highly proliferative T cells and may function as a stop signal for migration.

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Involvement of CD45 in early thymocyte development (2009)

CD45 is a protein tyrosine phosphatase that is expressed on all nucleated hematopoietic cells. The major substrates of CD45 in thymocytes and T cells are the Src family kinases Lck and Fyn. The role of CD45 in thymocyte development and T cell activation via its regulation of Src family kinases in T cell receptor signaling has been studied extensively. However, the role of CD45 in processes that affect thymocyte development prior to the expression of the T cell receptor has not been explored.The overall hypothesis of this study was that CD45 is a regulator of spreading, migration, proliferation, and differentiation of early thymocytes during development in the thymus and the absence of CD45 would alter the outcome of thymocyte development.The first aim was to determine how CD45 regulates CD44-mediated signaling leading to cell spreading. The interaction between CD44 and Lck was first examined. CD44 associated with Lck in a zinc-dependent and a zinc-independent manner. Mutation analysis localized the zinc-dependent interaction to the membrane proximal region of CD44, but did not involve individual cysteine residues on CD44. CD44 and Lck co-localized in microclusters upon CD44-mediated cell spreading. CD45 co-localized with Lck and CD44 in microclusters and with F-actin in ring structures. The recruitment of CD45 to microclusters may be a mechanism of how CD45 negatively regulates CD44-mediated spreading.The second specific aim was to determine the role of CD45 in migration, proliferation, and progression and differentiation of early thymocytes. CD45 negatively regulated CXCL12-mediated migration, and positively regulated the proliferation and progression of CD117- DN1 thymocytes. Absence of CD45 led to an altered composition of thymic subsets. The CD45-/- thymus contained decreased numbers of ETPs and an aberrant CD117- DN1 population that lacked CD24, TCRbeta, and CCR7 expression. There were also increased thymic NK and gamma/delta T cells, but decreased NKT cells. In addition, a novel intermediate between DN1 and DN2 that required Notch for progression was identified.Overall, this study identified new roles for CD45 in early thymocytes and provided a better picture of how the development of T cells, a central component of the immune system, is regulated.

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Determining a role for CD45 in dendritic cells (2008)

CD45 is a leukocyte specific protein tyrosine phosphatase present on the surface of all nucleated, hematopoietic cells. Despite its well-characterized role in antigen receptor signaling, little is known about its function in cell types like dendritic cells (DCs). DCs are crucial to the immune response both for its initiation and for its suppression. In this dissertation, the effects of the lack of CD45 on dendritic cell development and function were studied. The most important finding was that the lack of CD45 had a differential impact on the proinflammatory cytokine profiles elicited in DCs by different TLR agonists. TLR4 ligation led to a decrease in proinflammatory cytokine and IFNβ production whereas stimulation through TLR2 or TLR9 increased cytokine production. This suggests CD45 may be acting as a negative regulator of MyD88-dependent cytokine signaling and a positive regulator of the Trif pathway. The absence of CD45 caused alterations in the phosphotyrosine levels of several Src family kinases including Lyn. In CD45-/- DCs, Lyn was not activated upon LPS stimulation and several substrates of Lyn that appear as negative regulators in the MyD88-dependent pathway of TLR4 signaling are also not phosphorylated, providing evidence that CD45 may be a negative regulator of this pathway. The absence of CD45 in TLR activated DCs had an effect on the IFNγ secretion by CD4+ T cells and NK cells, consistent with the cytokine profiles of the DCs These data demonstrate that modulation of TLR signaling by CD45, in DCs, has the ability to impact the development of the adaptive immune response. The absence of CD45 in mice did not result in increased survival upon challenge with a high dose of LPS. Serum TNFα levels were increased in the CD45-/- mice and they showed more severe symptoms of septic shock. However, the CD45-/- mice were also found to have an increase in the number of peritoneal macrophages. Overall this study shows that CD45 does play an important role in cell types other than lymphocytes. CD45 is a regulator of TLR-mediated cytokine secretion in DCs and thus directs the outcome of the adaptive immune response.

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Regulation and function of hyaluronan binding by CD44 in the immune system (2008)

The proteoglycan CD44 is a widely expressed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, and is involved in processes ranging from metastasis to wound healing. In the immune system, leukocyte activation induces hyaluronan binding through changes in CD44 post-translational modification, but these changes have not been well characterized. Here I identify chondroitin sulfate addition to CD44 as a negative regulator of hyaluronan binding. Chondroitin sulfate addition was analyzed by sulfate incorporation and Western blotting and determined to occur at serine 180 in human CD44 using site-directed mutagenesis. Mutation of serine 180 increased hyaluronan binding by both a CD44-immunoglobulin fusion protein expressed in HEK293 cells, and full-length CD44 expressed in murine L fibroblast cells. In bone marrow-derived macrophages, hyaluronan binding induced by the inflammatory cytokines tumor necrosis factor-α and interferon-γ corresponded with reduced chondroitin sulfate addition to CD44. Retroviral infection of CD44⁻/⁻ macrophages with mouse CD44 containing a mutation at serine 183, equivalent to serine 180 in human CD44, resulted in hyaluronan binding that was constitutively high and no longer enhanced by stimulation. These results demonstrate that hyaluronan binding by CD44 is regulated by chondroitin sulfate addition in macrophages. A functional consequence of altered chondroitin sulfate addition and increased hyaluronan binding was observed in Jurkat T cells, which became more susceptible to activation-induced cell death when transfected with mutant CD44. The extent of cell death was dependent upon both the hyaluronan binding ability of CD44 and the size of hyaluronan itself, with high molecular mass hyaluronan having a greater effect than intermediate or low molecular mass hyaluronan. The addition of hyaluronan to pre-activated Jurkat T cells induced rapid cell death independently of Fas and caspase activation, identifying a unique Fas-independent mechanism for inducing cell death in activated cells. Results were comparable in splenic T cells, where high hyaluronan binding correlated with increased phosphatidylserine exposure, and hyaluronan-dependent cell death occurred in a population of restimulated cells in the absence of Fas-dependent cell death. Together these results reveal a novel mechanism for regulating hyaluronan binding and demonstrate that altered chondroitin sulfate addition can affect CD44 function.

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Master's Student Supervision

Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.

The role of the microenvironment and inflammation in the promotion of cancer metastasis (2018)

Circulating tumor cells become fully metastatic if they are able to extravasate from the microvasculature and move into microenvironmental niches within distant site organs where they survive and proliferate. To determine if inflammation facilitates this process, models of inflammatory asthma, hypersensitivity pneumonitis, or bleomycin-induced injury were used, followed by introduction of B16F0 melanoma cells into the microvasculature of the lungs. Strikingly, all three conditions increased overt metastasis without increasing extravasation and the number and size of early metastases were increased. Bleomycin induced inflammation led to the increased survival of B16F0 tumor cells and recruitment of monocyte derived macrophages (MoDM) to the lungs. These MoDM were located near the micrometastatic niche and their presence correlated with increased metastatic tumor cell burden. Inflammation also increased the deposition of the ECM component hyaluronan (HA) in the lung stroma and it was enriched in B16F0 containing metastatic nodules. HA binding through its cognate receptor CD44 correlates with an increase in the metastatic potential of B16 melanoma cells. However, deletion of CD44 using CD44-/- mice or CD44-/- B16F0 cells did not affect inflammation-driven increases in metastasis. Chondroitin sulphate (CS) was found to negatively regulate HA binding in B16 cells, and CS-null CD44 constitutively bound high levels of HA unlike parental B16F0 cells. Thus, high HA binding may be required for effects on metastasis, or HA may be priming the inflammatory premetastatic niche in a CD44 independent manner. Taken together these findings illustrate the importance of the microenvironment in distant site metastasis and they highlight inflammation as an important modifier of this microenvironment.

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The Impact of CD45 on Dendritic Cell Activiation and Tolerogenic Response (2012)

Dendritic cells (DCs) are potent antigen presenting cells that orchestrate the immune system to mediate either a pro- or anti-inflammatory response, by provision of critical instructive signals to T cells. The leukocyte-specific tyrosine phosphatase CD45 can influence the immune response by its ability to act as either a positive or negative regulator of DC pro-inflammatory cytokine production. This dissertation explores the effect of CD45 in mediating a tolerogenic response in DCs.The lack of CD45 in bone marrow derived dendritic cells (BMDCs) was found to cause a preferential production of IL-10 in response to LPS stimulation, despite the mature DC phenotype defined by elevated expression levels of MHCII and co-stimulatory molecules CD80 and CD86, and the unaffected ability of CD45 deficient BMDCs to drive naïve CD4⁺ T cells or CD4⁺ Foxp3⁺ regulatory T cell (Treg) proliferation in vitro. An important in vivo finding was that CD45 deficiency in RAG-/- mice provided greater protection against wasting disease in the Treg mediated prevention of experimental colitis. This survival advantage was found to correlate with an increased proportion of Tregs at the colonic lamina propria in CD45 deficient RAG-/- mice.Culture of BMDC precursors with the anti CD45RB antibody generated BMDCs with reduced LPS-induced IL-12 production and T cell stimulatory capacity implicating CD45RB expression in the promotion of tolerogenic responses in DCs. Rapamycin, a pharmacological inhibitor of the mTOR pathway, was found to have a minimal effect on inducing a tolerogenic DC from BMDCs generated by culture in GM-CSF alone. However rapamycin exhibited a more profound suppressive effect on the ability of CD45 deficient DCs to drive T cell proliferation in vitro.Overall this study suggests that the CD45RB isoform may specifically inhibit an inflammatory response in DCs but the loss of all CD45 isoforms in DCs may potentially mediate T tolerance through immune deviation, while the loss of CD45 in multiple innate immune cells may culminate in an environment that promotes Treg expansion or function in vivo.

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Publications

  • Splenic erythroid progenitors decrease TNF‐α production by macrophages and reduce systemic inflammation in a mouse model of T cell‐induced colitis (2021)
    European Journal of Immunology,
  • Regulation of CD71+TER119+ erythroid progenitor cells by CD45 (2020)
    Experimental Hematology,
  • RNA sequencing and lipidomic analysis of alveolar macrophages from normal and CD44 deficient mice (2019)
  • ATG Genes Influence the Virulence of Cryptococcus neoformans through Contributions beyond Core Autophagy Functions. (2018)
    Infection and immunity,
  • CD44-mediated hyaluronan binding marks proliferating hematopoietic progenitor cells and promotes bone marrow engraftment (2018)
    PLOS ONE,
  • CD44-mediated hyaluronan binding marks proliferating hematopoietic progenitor cells and promotes bone marrow engraftment. (2018)
    PloS one,
  • Hyaluronan and Its Interactions With Immune Cells in the Healthy and Inflamed Lung. (2018)
    Frontiers in immunology,
  • Hyaluronan-binding by CD44 reduces the memory potential of activated murine CD8 T cells (2018)
    European Journal of Immunology,
  • Hyaluronan-binding by CD44 reduces the memory potential of activated murine CD8 T cells. (2018)
    European journal of immunology,
  • Transmembrane Pickets Connect Cyto- and Pericellular Skeletons Forming Barriers to Receptor Engagement. (2018)
    Cell,
  • The survival of fetal and bone marrow monocyte-derived alveolar macrophages is promoted by CD44 and its interaction with hyaluronan. (2017)
    Mucosal immunology,
  • CD45 phosphatase is crucial for human and murine acute myeloid leukemia maintenance through its localization in lipid rafts (2016)
    Oncotarget, 7 (40), 64785
  • CD45 regulates GM-CSF, retinoic acid and T-cell homing in intestinal inflammation (2016)
    Mucosal immunology, 9 (6), 1514
  • Endotoxin free hyaluronan and hyaluronan fragments do not stimulate TNF-$α$, interleukin-12 or upregulate co-stimulatory molecules in dendritic cells or macrophages (2016)
    Scientific reports, 6, 36928
  • Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow (2016)
    JoVE (Journal of Visualized Experiments), (112), e54194
  • Hyaluronan binding identifies a functionally distinct alveolar macrophage--like population in bone marrow--derived dendritic cell cultures (2015)
    The Journal of Immunology, 195 (2), 632--642
  • The where, when, how, and why of hyaluronan binding by immune cells (2015)
    Frontiers in immunology, 6, 150
  • Arginase activity in alternatively activated macrophages protects PI3Kp110$δ$ deficient mice from dextran sodium sulfate induced intestinal inflammation (2014)
    European journal of immunology, 44 (11), 3353--3367
  • Innate immune cell CD45 regulates lymphopenia-induced T cell proliferation (2014)
    The Journal of Immunology, 193 (6), 2831--2842
  • Glycoproteins with Monoclonal Antibody Affinity Columns (2013)
    Hybridoma Technology in the Biosciences and Medicine, , 163
  • Temporal stability of the mouse gut microbiota in relation to innate and adaptive immunity (2013)
    Environmental microbiology reports, 5 (2), 200--210
  • CD45 (PTPRC) (2012)
    Encyclopedia of Signaling Molecules, , 328--334
  • Optical Methods (2012)
    The Enzymes of Biological Membranes: Volume 1 Membrane Structure and Dynamics, , 421
  • The tyrosine phosphatase CD45 regulates lymphopenia-induced T cell proliferation (47.15) (2012)
  • CD44-mediated elongated T cell spreading requires Pyk2 activation by Src family kinases, extracellular calcium, phospholipase C and phosphatidylinositol-3 kinase (2011)
    Cellular signalling, 23 (5), 812--819
  • Differential use of chondroitin sulfate to regulate hyaluronan binding by receptor CD44 in inflammatory and interleukin 4-activated macrophages (2011)
    Journal of Biological Chemistry, 286 (22), 19179--19190
  • Host defense peptide LL-37 selectively reduces proinflammatory macrophage responses (2011)
    The Journal of Immunology, 186 (9), 5497--5505
  • Hyaluronan binding identifies the most proliferative activated and memory T cells (2011)
    European journal of immunology, 41 (4), 1108--1119
  • The Suv39H1 methyltransferase inhibitor chaetocin causes induction of integrated HIV-1 without producing a T cell response (2011)
    FEBS letters, 585 (22), 3549--3554
  • CD44 interacts directly with Lck in a zinc-dependent manner (2010)
    Molecular immunology, 47 (10), 1882--1889
  • CD45 regulates migration, proliferation, and progression of double negative 1 thymocytes (2010)
    The Journal of Immunology, 185 (4), 2059--2070
  • Modulation of immune cell signalling by the leukocyte common tyrosine phosphatase, CD45 (2010)
    Cellular signalling, 22 (3), 339--348
  • Y-box binding protein-1 induces the expression of CD44 and CD49f leading to enhanced self-renewal, mammosphere growth, and drug resistance (2010)
    Cancer research, 70 (7), 2840--2851
  • CD44 and its role in inflammation and inflammatory diseases (2009)
    Inflammation & Allergy-Drug Targets (Formerly Current Drug Targets-Inflammation & Allergy), 8 (3), 208--220
  • CD45-mediated fodrin cleavage during galectin-1 T cell death promotes phagocytic clearance of dying cells (2009)
    The Journal of Immunology, 182 (11), 7001--7008
  • Tyrosine phosphorylation in immune cells: direct and indirect effects on toll-like receptor-induced proinflammatory cytokine production (2009)
    Critical Reviews� in Immunology, 29 (4)
  • CD45 Down-Regulates Lck-Mediated CD44 (2008)
    J Immunol, 181, 7033--7043
  • CD45 down-regulates Lck-mediated CD44 signaling and modulates actin rearrangement in T cells (2008)
    The Journal of Immunology, 181 (10), 7033--7043
  • CD45 regulates TLR-induced proinflammatory cytokine and IFN-$β$ secretion in dendritic cells (2008)
    The Journal of Immunology, 180 (12), 8020--8029
  • CD45 Regulates TLR-Induced Proinflammatory Cytokine and IFN-Secretion in Dendritic Cells (2008)
  • Hyaluronan induces cell death in activated T cells through CD44 (2008)
    The Journal of Immunology, 181 (10), 7044--7054
  • Differential function of PTP$α$ and PTP$α$ Y789F in T cells and regulation of PTP$α$ phosphorylation at Tyr-789 by CD45 (2007)
    Journal of Biological Chemistry, 282 (29), 20925--20932
  • Galectin-3 and galectin-1 bind distinct cell surface glycoprotein receptors to induce T cell death (2006)
    The Journal of Immunology, 176 (2), 778--789
  • H2-M3-restricted T cells participate in the priming of antigen-specific CD4+ T cells (2006)
    The Journal of Immunology, 177 (8), 5098--5104
  • Use of CD44 by CD4+ Th1 and Th2 lymphocytes to roll and adhere (2006)
    Blood, 107 (12), 4798--4806
  • Chondroitin sulfate addition to CD44H negatively regulates hyaluronan binding (2005)
    Biochemical and biophysical research communications, 334 (2), 306--312
  • Expression of CD45 lacking the catalytic protein tyrosine phosphatase domain modulates Lck phosphorylation and T cell activation (2005)
    Journal of Biological Chemistry, 280 (14), 14318--14324
  • Expression of N-acetylglucosamine 6-O-sulfotransferases (GlcNAc6STs)-1 and-4 in human monocytes: GlcNAc6ST-1 is implicated in the generation of the 6-sulfo N-acetyllactosamine/Lewis x epitope on CD44 and is induced by TNF-$α$ (2005)
    Glycobiology, 15 (7), 7C--13C
  • Regulation of hyaluronan binding by F-actin and colocalization of CD44 and phosphorylated ezrin/radixin/moesin (ERM) proteins in myeloid cells (2005)
    Experimental cell research, 303 (2), 400--414
  • Chondroitin sulfate chains on CD44 negatively regulate its ability to bind hyaluronan (2004)
    GLYCOBIOLOGY, 14 (11), 1108--1108
  • Role of CD44 and Hyaluronan in Neutrophil (2004)
    J Immunol, 173, 7594--7601
  • Role of CD44 and hyaluronan in neutrophil recruitment (2004)
    The Journal of Immunology, 173 (12), 7594--7601
  • Subdomain X of the kinase domain of Lck binds CD45 and facilitates dephosphorylation (2004)
    Journal of Biological Chemistry, 279 (5), 3455--3462
  • A cell death pathway induced by antibody-mediated cross-linking of CD45 on lymphocytes (2003)
    Critical Reviews� in Immunology, 23 (5-6)
  • Proteins and Proteomics Cumulative Contents (2003)
    Biochimica et Biophysica Acta, 1650, 147
  • The noncatalytic domains of Lck regulate its dephosphorylation by CD45 (2003)
    Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1650 (1-2), 40--49
  • Apoptosis mediated through CD45 is independent of its phosphatase activity and association with leukocyte phosphatase-associated phosphoprotein (2002)
    The Journal of Immunology, 168 (12), 6084--6089
  • CELLULAR IMMUNOLOGY AND IMMUNE REGULATION-Apoptosis Mediated Through CD45 Is Independent of Its Phosphatase Activity and Association with Leukocyte Phosphatase-Associated Phosphoprotein (2002)
    Journal of Immunology, 168 (12), 6084--6089
  • TNF-$α$ increases the carbohydrate sulfation of CD44: induction of 6-sulfo N-acetyl lactosamine on N-and O-linked glycans (2002)
    Glycobiology, 12 (10), 613--622
  • CD44-initiated cell spreading induces Pyk2 phosphorylation, is mediated by Src family kinases, and is negatively regulated by CD45 (2001)
    Journal of Biological Chemistry, 276 (31), 28767--28773
  • CD45 is a JAK phosphatase and negatively regulates cytokine receptor signalling (2001)
    Nature, 409 (6818), 349
  • MECHANISMS OF SIGNAL TRANSDUCTION-CD44-initiated cell spreading induces Pyk2 phosphorylation, is mediated by Src family kinases, and is negatively regulated by CD45. (2001)
    Journal of Biological Chemistry, 276 (31), 28767--28773
  • Role of sulfation in CD44-mediated hyaluronan binding induced by inflammatory mediators in human CD14+ peripheral blood monocytes (2001)
    The Journal of Immunology, 167 (9), 5367--5374
  • A role for the cell adhesion molecule CD44 and sulfation in leukocyte--endothelial cell adhesion during an inflammatory response? (2000)
    Biochemical pharmacology, 59 (5), 455--465
  • CD45 regulates CD CD44-initiated cytoskeletal changes and cell spreading in T cells (2000)
    FASEB JOURNAL, 14 (6), A1143--A1143
  • Characterization of the interaction between the tyrosine phosphatase, CD45, and its substrate, p56lck (2000)
    FASEB JOURNAL, 14 (6), A1174--A1174
  • Characterization of TNF-alpha induced sulfation of CD44 (2000)
    FASEB JOURNAL, 14 (6), A1142--A1142
  • Determination of CD45 tyrosine phosphatase activity in T lymphocytes (2000)
    T Cell Protocols, , 289--300
  • Regulation of hyaluronan binding by CD44 on human peripheral blood monocytes. (2000)
    FASEB JOURNAL, 14 (6), A1142--A1142
  • Stable interdomain interaction within the cytoplasmic domain of CD45 increases enzyme stability (2000)
    Biochemical and biophysical research communications, 271 (2), 292--298
  • Leukocyte adhesion mediated by sulfated CD44 (1999)
    Biomedicine & Pharmacotherapy, 8 (53), 393
  • Analysis of CD44 interactions with hyaluronan in murine L cell fibroblasts deficient in glycosaminoglycan synthesis: a role for chondroitin sulfate (1998)
    Journal of Cell Science, 111 (7), 1021--1029
  • CD45: A family of leukocyte-specific cell surface glycoproteins (1998)
    Weir's handbook of experimental immunology, 2, 62--1
  • Characterization of Recombinant CD45 Cytoplasmic Domain Proteins EVIDENCE FOR INTRAMOLECULAR AND INTERMOLECULAR INTERACTIONS (1998)
    Journal of Biological Chemistry, 273 (28), 17839--17845
  • Chimeric CD4/CD44 molecules associate with CD44 via the transmembrane region and reduce hyaluronan binding in T cell lines (1998)
    European journal of immunology, 28 (6), 1745--1754
  • PROTEIN CHEMISTRY AND STRUCTURE-Characterization of recombinant CD45 cytoplasmic domain proteins. Evidence for intramolecular and intermolecular interactions. (1998)
    Journal of Biological Chemistry, 273 (28), 17839--17845
  • Regulation of CD44 mediated adhesion by sulfation (1998)
    MOLECULAR BIOLOGY OF THE CELL, 9, 198A--198A
  • The association between CD20 and Src-family tyrosine kinases requires an additional factor (1998)
    Molecular immunology, 35 (4), 207--214
  • TNF-$α$ induction of CD44-mediated leukocyte adhesion by sulfation (1998)
    Science, 282 (5390), 941--943
  • CD45 and RPTP$α$ display different protein tyrosine phosphatase activities in T lymphocytes (1997)
    Biochemical Journal, 327 (3), 867--876
  • Demonstration of a Direct Interaction between p56 and the Cytoplasmic Domain of CD45 in Vitro (1996)
    Journal of Biological Chemistry, 271 (3), 1295--1300
  • Increase in the specific activity of p50csk in proliferating T cells correlates with decreased specific activity of p56lck and p59fyn and reduced phosphorylation of CD3 subunits (1996)
    Molecular immunology, 33 (6), 531--540
  • Regulation of hyaluronan binding to CD44 by chondroitin sulfate and N-glycosylation. (1996)
    MOLECULAR BIOLOGY OF THE CELL, 7, 374--374
  • Association of 75/80-kDa Phosphoproteins and the Tyrosine Kinases Lyn, Fyn, and Lck with the B Cell Molecule CD20 EVIDENCE AGAINST INVOLVEMENT OF THE CYTOPLASMIC REGIONS OF CD20 (1995)
    Journal of Biological Chemistry, 270 (38), 22632--22638
  • Characterization of the interaction between CD45 and CD45-AP (1995)
    Journal of Biological Chemistry, 270 (36), 21151--21157
  • Detection of restricted isoform expression and tyrosine phosphatase activity of CD45 in murine dendritic cells (1995)
    European journal of immunology, 25 (12), 3370--3374
  • Non-radioactive method to measure CD45 protein tyrosine phosphatase activity isolated directly from cells (1995)
    Journal of immunological methods, 179 (2), 177--185
  • Point mutation in the second phosphatase domain of CD45 abrogates tyrosine phosphatase activity (1995)
    Biochemical and biophysical research communications, 206 (1), 302--309
  • Isoforms of the transmembrane tyrosine phosphatase CD45 differentially affect T cell recognition (1994)
    Immunity, 1 (2), 109--119
  • Protein tyrosine phosphorylation in Mycobacterium tuberculosis (1994)
    FEMS microbiology letters, 124 (2), 203--207
  • Specific CD45 isoforms differentially regulate T cell receptor signaling. (1994)
    The EMBO journal, 13 (4), 798--807
  • Constitutive tyrosine phosphorylation of the T-cell receptor (TCR) zeta subunit: regulation of TCR-associated protein tyrosine kinase activity by TCR zeta. (1993)
    Molecular and cellular biology, 13 (9), 5771--5780
  • Expression of variable exon A-, B-, and C-specific CD45 determinants on peripheral and thymic T cell populations. (1992)
    The Journal of Immunology, 148 (1), 19--28
  • Mutational analysis of CD45. A leukocyte-specific protein tyrosine phosphatase. (1992)
    Journal of Biological Chemistry, 267 (12), 8035--8041
  • Regulation and structure-function relationship of CD45 (1992)
  • Structure and function of CD45: a leukocyte-specific protein tyrosine phosphatase (1992)
    Mechanisms of Lymphocyte Activation and Immune Regulation IV, , 29--37
  • CD45-A LEUKOCYTE-SPECIFIC PROTEIN TYROSINE-PHOSPHATASE (1991)
    FASEB JOURNAL, 5 (4), A784--A784
  • CD45: a leukocyte-specific member of the protein tyrosine phosphatase family (1991)
    Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1095 (1), 46--56
  • Differential expression of the alternatively spliced exons of murine CD45 in Th1 and Th2 cell clones (1991)
    European journal of immunology, 21 (1), 17--22
  • Changes in CD45 isoform expression accompany antigen-induced murine T-cell activation (1989)
    Proceedings of the National Academy of Sciences, 86 (17), 6734--6738
  • Expression of CD45 alters phosphorylation of the lck-encoded tyrosine protein kinase in murine lymphoma T-cell lines (1989)
    Proceedings of the National Academy of Sciences, 86 (22), 8959--8963
  • Identification of the alternatively spliced exons of murine CD45 (T200) required for reactivity with B220 and other T200-restricted antibodies. (1989)
    Journal of Experimental Medicine, 169 (3), 1179--1184
  • CD8B, the human equivalent of the mouse Ly-3 gene is localized on chromosome 2 (1988)
    Immunogenetics, 27 (1), 70--72
  • Immunoglobulin-related structures associated with vertebrate cell surfaces (1988)
    The T-Cell Receptors, , 53--87
  • A human homolog of the mouse CD8 molecule, Lyt-3: genomic sequence and expression (1987)
    Immunogenetics, 26 (3), 174--177
  • CD8 THE HUMAN EQUIVALENT OF THE MOUSE LYT-3 GENE IS ASSIGNED TO CHROMOSOME-2 (1987)
    CYTOGENETICS AND CELL GENETICS, 46 (1-4), 698--698
  • T4. 5 Identification of human Lyt 3 at the genomic level (1987)
    Leucocyte Typing III: White Cell Differentiation Antigens, , 215
  • Human Hybridomas and Monoclonal Antibodies (1986)
  • Purification and sequence of the rat CDS antigen, a cytotoxic T-cell marker (1986)
  • Striking similarities between antigen receptor J pieces and sequence in the second chain of the murine CD8 antigen (1986)
    Nature, 323 (6083), 74
  • I. HISTORICAL BACKGROUND (1985)
    The Enzymes of Biological Membranes: Membrane structure and dynamics, 1, 421
  • Optical methods for measuring the rotational diffusion of membrane proteins (1985)
    Spectroscopy and the dynamics of Molecular Biological Systems, PM Bayley and RE Dale, eds, , 95--118
  • Purification of membrane glycoproteins with monoclonal antibody affinity columns (1985)
    Hybridoma Technology in the Biosciences and Medicine, , 163--175
  • Purification, chain separation and sequence of the MRC OX-8 antigen, a marker of rat cytotoxic T lymphocytes. (1985)
    The EMBO journal, 4 (10), 2539--2545
  • Rotational diffusion of membrane proteins optical methods (1985)
    The Enzymes of Biological Membranes, , 421--439
  • High affinity epidermal growth factor receptors on the surface of A431 cells have restricted lateral diffusion. (1984)
    The EMBO journal, 3 (8), 1843--1847
  • Carbocyanine dyes used as fluorescent triplet probes for measuring slow rotational diffusion of lipids in membranes (1983)
    FEBS letters, 153 (2), 391--394
  • Intracellular pH and free calcium changes in single cells using quene 1 and quin 2 probes and fluorescence microscopy (1983)
    FEBS letters, 161 (1), 21--27
  • Changes in the properties of the surface membrane of Schistosoma mansoni during growth as measured by fluorescence recovery after photobleaching (1982)
    FEBS letters, 141 (1), 132--135
  • Fluorescent triplet probes for measuring the rotational diffusion of membrane proteins (1982)
    Biochemical Journal, 203 (1), 313--321
  • Depolarization of fluorescence depletion: A microscopic method for measuring rotational diffusion of membrane proteins on the surface of a single cell (1981)
    FEBS letters, 132 (2), 252--256
 
 

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