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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - May 2019)
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.
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.
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.
No abstract available.
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.
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.
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.
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.
Master's Student Supervision (2010 - 2018)
No abstract available.
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.
- CD44-mediated hyaluronan binding marks proliferating hematopoietic progenitor cells and promotes bone marrow engraftment (2018)
- Hyaluronan-binding by CD44 reduces the memory potential of activated murine CD8 T cells (2018)
European Journal of Immunology,