Melinda Ekkens
Molecular and Cell Biology Program
Doctor of
Philosophy
2002
Major Advisor: William C. Gause, Ph.D., Department of Microbiology & Immunology
Thesis Title: The Role of Costimulatory Molecules in the Development of Memory and Effector T helper 2 Cells During an in vivo Immune Response to the Murine Gastrointestinal Parasite Heligmosomoides polygyrus
ABSTRACT
Previous studies have demonstrated the importance of costimulatory interactions for effector CD4+ T helper (Th) cell development during the primary immune response. However, the role of costimulatory molecules in memory CD4+ T cell differentiation is not well understood. One model used to study the Th immune response involves oral infection of mice with the gastrointestinal nematode parasite Heligmosomoides polygyrus. Although the primary immune response to H. polygyrus is a chronic infection, challenge immunization triggers a T-dependent memory response that impairs adult worm maturation. In the studies presented herein, the effects of costimulatory molecule blockade on T helper effector cell function during the memory response were examined.
Effector T cell development was inhibited during the primary response to H. polygyrus in B7-1/B7-2-/- mice; however, memory Th cells developed that produced IL-4 and mediated effective reductions in adult worm egg production, but did not provide effective Ag-specific B cell help or support increased germinal center (GC) formation. Parallel studies in H. polygyrus-challenged CD28-/- mice demonstrated similar IL-4 elevations and decreases in adult worm egg production. However, Ag-specific Ab responses and increased GC formation were significantly restored in H. polygyrus-inoculated CD28-/- mice.
Although elevations in serum IgG1 and GC formation were intact in H. polygyrus-challenged OX40L-/- mice, elevations in IL-4 and serum IgE were partially inhibited, and associated with decreased worm expulsion and increased egg production. To further examine the role of OX40L in Ag-specific CD4+ T cell IL-4 production following priming, adoptively transferred OVA-specific DO11.10 T cells were analyzed in the context of the H. polygyrus response. Following immunization with OVA plus H. polygyrus, Ag-specific T cell expansion, cell cycle progression, CXCR5 expression, and migration were comparable in OX40L+/+ and OX40L-/- mice; however, Ag-specific T cell IL-4 production was reduced in OX40L-/- mice, suggesting a preferential role for OX40L costimulation in IL-4 production.
These studies extend our understanding of the role of costimulatory molecules in the development of memory Th2 cells during infectious disease. They also suggest that B7-1/B7-2 antagonists may be particularly effective in the treatment of chronic diseases where continuous renewal of effector populations from naïve precursor T cells mediates pathogenesis.
Seth Ettenberg
Molecular and Cell Biology
Doctor of
Philosophy
2001
Major Advisor: Stan Lipkowitz, M.D., Ph.D. Molecular and Cell Biology Program
Title of Thesis: Regulation of Epidermal Growth Factor Receptor Signaling by chi-b
Abstract
Negative regulation of the Epidermal Growth Factor Receptor (EGFR) signaling is essential to proper regulation of cellular function. Genetic evidence from C. elegans and Drosophila melanogaster has demonstrated that cbl proteins are negative regulators of the EGFR in these organisms. To determine the role of mammalian cbl proteins in EGFR signaling, stable clones overexpressing cbl-b were created in two cell lines which have distinct biological responses to EGFR activation. In the 32D/EGFR murine hematopoetic cell line, overexpression of cbl-b inhibits Epidermal Growth Factor- induced (EGF) proliferation. In the MDA-MB-468 human breast cancer cell line, EGFR activation induces apoptosis. Overexpression of cbl-b in these cells inhibits EGF-induced-apoptosis. These data demonstrate that the mammalian cbl-b protein, like the C. elegans and Drosophila homologs, inhibits EGFR function. The molecular basis of this inhibition was studied in these two model systems. cbl-b is phosphorylated and recruited to the EGFR upon activation. In both cell lines, activation of the EGFR and activation of multiple downstream pathways have a shortened duration of signaling when cbl-b is overexpressed. Further biochemical analysis demonstrated that cbl-b increased activation-induced downregulation of the EGFR by enhancing EGFR ubiquitination and EGFR degradation. Specific inhibitors of either lysosomal or proteasomal proteases blocked cbl-b mediated EGFR degradation. Further analysis of cbl-b expression after cells are stimulated with EGF demonstrated that cbl-b is coordinately degraded along with the EGFR. Both EGFR and cbl-b downregulation requires an intact Tyrosine Kinase Binding and RING finger domain of the cbl-b protein. Additionally, binding of cbl-b to the EGFR is required for either protein to undergo EGF-induced degradation. Other proteins which are recruited to the activated EGFR complex are also coordinately degraded with the EGFR and cbl-b. These data allow us to construct a model of the regulation of the EGFR by cbl-b. Upon activation of the EGFR, cbl-b is phosphorylated and recruited to the EGFR. Next cbl-b enhances ubiquitination and subsequent degradation of the EGFR and other proteins bound to it. Finally, these data demonstrate that the mammalian protein cbl-b, like the C. elegans and Drosophila homologs, inhibits EGFR function.
Agnes Y. Jones-Trower
Molecular and Cell Biology Program
Doctor of
Philosophy
2001
Major Advisor: Christopher C. Broder, Ph.D., Department of Microbiology and Immunology
Thesis Title: Characterization and Enhanced Processing of Soluble, Oligomeric gp140 Envelope Glycoproteins Derived from Human Immunodeficiency Virus Type-1 Primary Isolates
ABSTRACT
HIV-1 first interacts with host cells through its envelope glycoprotein (Env), the major target of neutralizing antibodies. The biologically relevant form of Env is an oligomer, both in its presentation to the immune system and in virus entry. Also, native Env is cleaved into its gp120 and gp41 non-covalently associated subunits. Env-based vaccines have thus far failed to efficiently generate broadly cross-reactive, neutralizing antibodies towards primary isolates. Possible explanations for their failure are either they were derived from laboratory-adapted strains or they have not preserved the conformational structures necessary to elicit broadly reactive responses. Producing a broadly effective Env-based vaccine is also potentially complicated by the existence of multiple HIV-1 genotypes. Here, I have constructed a panel of truncated env genes from primary isolates of several different HIV-1 clades. Recombinant vaccinia viruses expressing these genes produce a secreted Env known as gp140. These gp140s were characterized by sucrose density gradient centrifugation and size exclusion chromatography analyses to determine oligomeric status and degree of processing. While most processed gp140s dissociated to monomeric forms, there was evidence that certain isolates could retain gp120 in an oligomer. A large scale purification scheme was developed using lentil lectin affinity and size exclusion chromatographies to prepare gp140 oligomers. Pre- and post-production processing enhancers (furin and plasmin) were examined as means to bolster the amount of processed gp140, and reducible crosslinkers were employed to analyze the ability of processed gp140 to maintain oligomeric forms. The antigenic properties of the gp140s, both cleaved vs. uncleaved, and crosslinked vs. non-crosslinked, were analyzed by immunoprecipitation with a panel of well-characterized human and mouse antibodies. The purified, crosslinked, cleaved oligomers retained important epitopes and the ability to undergo receptor-induced conformational change as could uncleaved gp140. Finally, a novel antigen delivery system (Matrix III) was investigated where oligomeric gp140 was encapsulated and administered to animals. Analysis of these sera has indicated that Matrix III does not seem to alter the quantity or quality of elicited antibody. Taken together, these experiments have provided critical information and material towards testing a variety of oligomeric Env-based vaccine strategies for the prevention of HIV-1 infection.
Velia Mitro
Molecular and Cell Biology Program
Doctor of
Philosophy
2000
Major Advisor: Dr. William C. Gause, Department of Microbiology and Immunology
Thesis Title: The function of CTLA4 during the in vivo immune response to infectious disease.
ABSTRACT
CD4+ T cells playa key role in the adaptive immune response to foreign antigens. For T cells to be activated, two signals are required. The first signal is delivered through antigen recognition by the T cell receptor. A second, or costimulatory, signal is also required for optimal activation of T cells. CD28 ligation by B7 is a potent mediator of positive costimulation. In contrast, B7 ligation of CTLA4 (CD152), a homologue of CD28, provides a critical downregulatory signal. Recent data has suggested that CTLA4 may also share some stimulatory functions with CD28. Because costimulatory molecule interactions are critical for many immune responses, a greater understanding of CTLA4 function may promote development of immunotherapies where enhancement or inhibition of the immune response would be clinically beneficial. This research was directed at developing a greater understanding of CTLA4 function in the immune response to infectious disease. A murine model of gastrointestinal nematode infection, Heligmosomoides polygyrus, was utilized in this research to investigate the role of CTLA4 after onset of infection, once naive T cells have differentiated to effector T cells. These data support a negative regulatory role for CTLA4 late in the response. Blockade of CTLA4 by in vivo administration of anti-CTLA4 antibody enhanced the polarized Th2 response to H polygyrus, resulting in increased serum concentrations of immunoglobulins, IL-4 secretion, and T and B cell activation. Further evidence of enhanced immune response upon CTLA4 blockade was provided in another nematode model, Trichuris muris. Anti-CTLA4 antibody treatment increased serum immunoglobulin concentrations and T and B cell activation. The treatment also caused immune deviation from Th1 to Th2, as evidenced by decreased IFNm and increased IL-4 secretion. These data are consistent with a model for Th1 versus Th2 cell differentiation which describes the decision as based on the balance between strength of signal and innate response. On a molecular level, the phosphorylation events following CTLA4 blockade were examined, and intracellular binding partners for CTLA4 and CD28 were identified. The dependence of effector T cells upon continued combined CTLA4/CD28 signaling was also explored.
Joseph A.
Nielsen
Molecular and Cell Biology
Doctor of
Philosophy
2002
Major Advisor:
Regina Armstrong, Ph.D., Department of Anatomy, Physiology
and Genetics
Thesis Title:Nuclear Organization and Myt1 Interaction in Transcriptional Control of Neural Cell Differentiation
Abstract
Neural
cell differentiation is a complex set of events beginning with cells responding
to both soluble and cell contact-dependent external signals. These signals
activate pathways that lead to changes in gene expression patterns, which
ultimately give rise to the differentiated cell phenotype. In these studies,
potential mechanisms regulating different aspects of oligodendrocyte differentiation
were explored. Specifically, these studies examined the contribution of gene
and protein localization to the establishment and/or maintenance of terminally
differentiated oligodendrocyte gene expression patterns and the role of myelin
transcription factor 1 (Myt1) in the regulation of oligodendrocyte proliferation
and differentiation.
Myt1 is a zinc-finger DNA-binding protein that is expressed in neural progenitors
and is localized to discrete domains within the nucleus of oligodendrocyte
progenitors. Primary oligodendrocyte lineage cells were examined during cell
differentiation in order to study the localization of the highly expressed
tissue-specific proteolipid protein gene relative to nuclear proteins such
as Myt1 and splicing factors within interphase nuclei. These data support
a nuclear organization model in which nuclear proteins and genes exhibit specific
patterns of distribution within nuclei, and activation of tissue-specific
genes is associated with changes in protein distribution rather than changes
in gene localization.
Myt1 contains six zinc-finger DNA-binding domains with sets of two N-terminal and of four C-terminal zinc-fingers. A retroviral expression system was used to overexpress the four zinc-finger DNA-binding domain of Myt1 (4FMyt1) which lacks the putative domains for protein-protein interaction and transcriptional activation. In a dominant negative study, expression of 4FMyt1 inhibited both proliferation and differentiation of oligodendrocyte progenitors. These data indicate that Myt1 contributes to the regulation of oligodendrocyte lineage development in the transitional period between proliferating progenitor cells and terminally differentiated oligodendrocytes.
These studies demonstrate the importance of Myt1 and nuclear organization to the regulation of oligodendrocyte progenitor differentiation and the establishment of tissue-specific gene expression patterns.
John T. Pesce
Molecular and Cell Biology Program
Doctor of Philosophy
2006
Major Advisor: William Gause, Ph.D., Department of Microbiology & Immunology
Thesis Title: Early Events Leading to the Host Protective Th2
ABSTRACT
Events necessary in the development of Th2 immune responses are poorly understood. A popular model used to study the development of these responses involves intracutaneous inoculation with the intestinal nematode parasite Nippostrongylus brasiliensis. Using B7-1/B7-2-/- mice infected with N. brasiliensis, we have shown that Th2 effector cells are capable of developing in the absence of B7 signaling interactions, although a substantial decrease in B cell Ag-specific Ab production was observed. To examine the mechanism of T cell activation, OVA-specific DO11.10 T cells were transferred to recipient mice, which were then immunized with a combination of N. brasiliensis plus OVA or either alone. Only the combination of N. brasiliensis plus OVA triggered T cell differentiation to OVA-specific Th2 cells, suggesting that N. brasiliensis acts as an adjuvant to stimulate Ag-specific naive T cells to differentiate to effector Th2 cells.
The adjuvant-like properties of N. brasiliensis suggested an
innate component of the immune response may be involved in Th2 development.
Using microarray analysis, draining ear lymph nodes from N. brasiliensis infected
mice exhibited significant increases in CCL2 which is known to be involved
in the recruitment of Gr-1+ neutrophils. Flow cytometric and immunofluorescent
analysis of infected lymph nodes resulted in the observation of an increased
presence of Gr-1+ cells. Depletion experiments, using anti-Gr-1 Ab, resulted
in disruption of the polarized Th2 in vivo immune response, characterized
by significantly increased levels of IFN-? gene expression, IgG2a elevations,
and increased worm burden. CCL2-/- deficient mice infected with N. brasiliensis
were used to determine if CCL2/CCR2 interactions were required for Gr-1 recruitment.
CCL2 deficiency resulted in significantly decreased Gr-1bright cell recruitment.
Absence of this population had an effect similar to that observed in anti-Gr-1
treatment experiments with increases in IFN-? and Th1 associated immunoglobulins.
Flow cytometric sorting and mRNA analysis of Gr-1bright cells revealed that
they consist of a purely neutrophil population which expresses high levels
of TNF-a and TGF-ß. These studies show the integral role that the innate
immune response plays in the development of a highly polarized Th2
immune response.
Overall, these studies have made significant contributions to
the understanding of the development of Th2 immune responses. The adaptation
of DO11.10 system into a Th2 context provides an essential tool which will
allow the determination of specific factors that result in the activation
of naïve T cells. As a direct result of developing this tool, we identified
a neutrophil population that is essential for the proper polarization of Th2
responses. This finding is quite significant in that this is the first time
that a neutrophil population has been implicated in the development of a Th2
immune response. While this work is still in its infancy, the work detailed
in this thesis provides evidence that neutrophils may prove to be a significant
target for future drug interventions in the field of allergy and asthma.
Sara K. Snyder
Molecular and Cell Biology
Doctor of
Philosophy
2000
Major Advisor: Gabriela Dveksler, Molecular and Cellular Biology Program
Thesis Title: Human Pregnancy-Specific Glycoproteins Function as lmmunomodulators In Vitro by Inducing Secretion of IL-10 and IL-6 in Human Monocytes
ABSTRACT
The lack of rejection of the semiallogeneic fetus by the maternal immune system is brought about in part by the maintenance of an anti-inflammatory immune environment at the maternal-fetal interface. The fetoplacental unit produces an arrray of cytokines and other regulatory molecules that assist in the implantation, survival and development of the fetus. Pregnancy specific glycoproteins (PSGs) are a family of highly conserved, secreted proteins abundantly produced by the placenta in various species including human, mouse and rat. PSGs are composed of repeated immunoglobulin (Ig) related domains, and are part of the Ig superfamily. Abnormally low levels of PSGs in maternal serum have been correlated with complications of pregnancy including spontaneous abortion. A peptide derived from the N-terminal domain of human PSG11 has been shown to bind cells of the promonocyte lineage, suggesting a role for PSGs in modulation of macrophage function during pregnancy.
We
investigated the ability of three recombinant human PSGs (PSG 1, PSG6 and
PSG 11 ), produced using a baculovirus expression system, to regulate the
in vitro production of cytokines by human monocytes. Cytokine secretion by
monocytes at 24 hours after treatment was measured by quantitative sandwich
ELISA. All three PSGs induced dose-dependent secretion of IL-10and IL-6, but
not secretion of TNF-", IL-I$ or IL-12. In order to examine the role
of the N-terminal Ig-variable-like domain in PSG function, we produced a fusion
protein consisting of only the N-terminal domain of PSG6. The PSG6 N-terminal
domain was shown to be sufficient for induction of monocyte secretion of IL-I
0 and IL-6, demonstrating that this domain mediates the interaction with a
putative PSG receptor on monocytes. As shown by RT-PCR, increased IL-10 and
IL-6 secretion was accompanied by an increase in mRNA after PSG6 treatment.
PSG6 induction of IL-I 0 and IL-6 secretion was inhibited by the tyrosine
kinase inhibitor Herbimycin A, the protein kinase C inhibitor Calphostin C,
and the specific protein kinase A inhibitor (Rp)cAMPS, suggesting a possible
role for these intracellular signalling molecules in PSG signal transduction
in monocytes. Also, the specific phosphodiesterase type IV inhibitor and cAMP
elevating agent, rolipram, increased monocyte secretion of IL-10 and IL-6
after treatment with PSG6, indicating that increased production of these cytokines
in response to PSGs may be mediated by an increase in cAMP. We also showed
that PSGs exhibit cross-species activity in cytokine induction using human
PSG treatment of a mouse macrophage cell line, RAW 264.7, and mouse PSG18
N-domain protein treatment of human monocytes, indicating that PSG function
may be highly conserved between species. Our results are consistent with a
role for PSGs in modulation of macrophage inflammatory responses at the maternal-
fetal interface where PSGs are in high concentration.