138 research outputs found
The Role of Mechanism of Action of CD200:CD200R1 Interaction in Breast Cancer
No full textCancer cells can use immune inhibitory receptors to evade the host’s anti-tumour responses and establish immunosuppressive networks in the tumour microenvironment. In this thesis, we investigated the interaction between the immunosuppressive molecule CD200 with its receptor, CD200R1, in breast cancer. We found that CD200 is expressed in the local tumour microenvironment in human breast cancer patients and developed a mouse model to study the effect of CD200 on tumour growth and metastasis. Using mouse-derived EMT6 breast cancer cells and BALB/c female hosts, we explored the effects of overexpressing and silencing the expression of CD200 and CD200R1 in hosts and tumour cells.
CD200 expression by host and tumour cells enhanced tumour growth and metastasis to draining lymph nodes (DLN). Silencing CD200 expression in EMT6 tumour cells led to a reduction in primary tumour size and metastasis, as well as an increase in anti-tumour cytotoxic responses in the host. Lack of CD200R1 expression in the host resulted in a marked decrease in breast cancer development and CD200R1-/- mice were able to mount specific anti-EMT6 immune response that could be adoptively transferred to wild type naïve hosts. In addition, we extended our findings to a model in which anti-tumour immunity was explored in EMT6 tumour-bearing hosts lacking CD200 expression and treated with a combination of immunotherapy with the non-conventional chemotherapeutic agent, metformin. The findings suggest that CD200 may be an important prognostic marker and a target for breast cancer treatment that could synergize with other therapies and improve outcomes in patients.Ph.D
CD200: A Novel Therapeutic Target for Chronic Lymphocytic Leukemia
No full textThe ability of cancer cells to escape anti-tumor immune responses is acknowledged as one of the hallmarks of cancer. Overexpression of immunoregulatory molecules is one mechanism responsible for the immunsuppressive network that is characteristic of the tumor microenvironment.
In this thesis, we investigated the role of CD200, a potent immunoregulatory molecule, in Chronic Lymphocytic Leukemia. We showed that functional blockade of CD200 on lymphoma cells or primary CLL cells, both of which express CD200 at high levels, augmented cytotoxic killing of these cells by effector CD8+ T cells in vitro. We also identified and characterized a previously unrecognized soluble form of CD200, sCD200, present in elevated levels in CLL plasma when compared to plasma from controls.
The data reported show that patients with high sCD200 levels have more aggressive disease, inferring that sCD200 may be a novel prognostic marker for CLL. The in vivo function of sCD200 was investigated for its ability to support engraftment of CLL splenocytes in NOD.SCID mice. Infusion of sCD200hi CLL plasma, but not sCD200lo normal plasma, enhanced engraftment of CLL-splenocytes in vivo, an effect which was abrogated by depletion of sCD200 from CLL plasma. The prolonged engraftment of CLL cells seen in this model (>6 months) suggests these mice represent a useful pre-clinical model for drug screening. The effect of CD200 blockade was tested in this model, and was found to be as effective in eliminating engrafted CLL cells as rituximab. Investigation of the mechanisms leading to the release of sCD200 from CLL cells showed that sCD200 was produced following ectodomain shedding by ADAM proteases and MMPs.
Results from studies reported in this thesis support the hypothesis that CD200 plays a major role in CLL biology, and suggests it may represent a novel therapeutic target for CLL.Ph
The Role of Mechanism of Action of CD200:CD200R1 Interaction in Breast Cancer
No full textCancer cells can use immune inhibitory receptors to evade the host’s anti-tumour responses and establish immunosuppressive networks in the tumour microenvironment. In this thesis, we investigated the interaction between the immunosuppressive molecule CD200 with its receptor, CD200R1, in breast cancer. We found that CD200 is expressed in the local tumour microenvironment in human breast cancer patients and developed a mouse model to study the effect of CD200 on tumour growth and metastasis. Using mouse-derived EMT6 breast cancer cells and BALB/c female hosts, we explored the effects of overexpressing and silencing the expression of CD200 and CD200R1 in hosts and tumour cells.
CD200 expression by host and tumour cells enhanced tumour growth and metastasis to draining lymph nodes (DLN). Silencing CD200 expression in EMT6 tumour cells led to a reduction in primary tumour size and metastasis, as well as an increase in anti-tumour cytotoxic responses in the host. Lack of CD200R1 expression in the host resulted in a marked decrease in breast cancer development and CD200R1-/- mice were able to mount specific anti-EMT6 immune response that could be adoptively transferred to wild type naïve hosts. In addition, we extended our findings to a model in which anti-tumour immunity was explored in EMT6 tumour-bearing hosts lacking CD200 expression and treated with a combination of immunotherapy with the non-conventional chemotherapeutic agent, metformin. The findings suggest that CD200 may be an important prognostic marker and a target for breast cancer treatment that could synergize with other therapies and improve outcomes in patients.Ph.D
CD200: A Novel Therapeutic Target for Chronic Lymphocytic Leukemia
No full textThe ability of cancer cells to escape anti-tumor immune responses is acknowledged as one of the hallmarks of cancer. Overexpression of immunoregulatory molecules is one mechanism responsible for the immunsuppressive network that is characteristic of the tumor microenvironment.
In this thesis, we investigated the role of CD200, a potent immunoregulatory molecule, in Chronic Lymphocytic Leukemia. We showed that functional blockade of CD200 on lymphoma cells or primary CLL cells, both of which express CD200 at high levels, augmented cytotoxic killing of these cells by effector CD8+ T cells in vitro. We also identified and characterized a previously unrecognized soluble form of CD200, sCD200, present in elevated levels in CLL plasma when compared to plasma from controls.
The data reported show that patients with high sCD200 levels have more aggressive disease, inferring that sCD200 may be a novel prognostic marker for CLL. The in vivo function of sCD200 was investigated for its ability to support engraftment of CLL splenocytes in NOD.SCID mice. Infusion of sCD200hi CLL plasma, but not sCD200lo normal plasma, enhanced engraftment of CLL-splenocytes in vivo, an effect which was abrogated by depletion of sCD200 from CLL plasma. The prolonged engraftment of CLL cells seen in this model (>6 months) suggests these mice represent a useful pre-clinical model for drug screening. The effect of CD200 blockade was tested in this model, and was found to be as effective in eliminating engrafted CLL cells as rituximab. Investigation of the mechanisms leading to the release of sCD200 from CLL cells showed that sCD200 was produced following ectodomain shedding by ADAM proteases and MMPs.
Results from studies reported in this thesis support the hypothesis that CD200 plays a major role in CLL biology, and suggests it may represent a novel therapeutic target for CLL.Ph
Mechanism(s) of Action of the Novel Immunoregulatory Molecule, CD200
No full textBoth CD200 and its receptor(s), CD200R(s), are type I membrane glycoproteins belonging to the immunoglobulin (Ig) supergene family. CD200:CD200R(s) interaction manipulates host immunity in multiple models, including those exploring allograft rejection, autoimmune disease, tumor development, spontaneous fetal loss, infection/inflammation, and virus infection. The studies described in this thesis were focused on investigation possible mechanism(s) involved in CD200-mediated regulation, using transgenic mice over-expressing CD200, and exploring models of skin allograft rejection and LPS-induced abortion in mice.
A Tet-on system was chosen to create CD200tg mice (rtTA CD200tg animal line), in which transgenic expression of CD200 is induced by the presence of doxycycline (Dox-treated mice). Splenocytes from Dox-treated transgenic mice, used as either responder cells or stimulator cells in mixed leukocyte cultures, showed antigen-specific suppressed lymphocyte proliferation and induction of CTL. Although enhanced survival of skin allografts was achieved in Dox-treated transplanted CD200tg mice (BALB/c to BL/6 Tg), all grafts were rejected by 28 days post transplantation (see chapter 2).
A superior “second generation” Tet-on system was used to create a new CD200tg animal referred to as rtTA2s-M2 CD200tg mice. Transgenic overexpression of CD200 in this mouse was stably induced at much lower Dox concentrations, with less (or no) “leaky” expression of the transgene in the absence of Dox. Using these mice in an LPS-induced murine abortion model, transgenic expression of CD200 was found to reduce the LPS-induced abortion rate from ~49% to 6% (see chapter 3).
Long term increased survival of grafted tissues (of both cardiac and skin allografts) was achieved using the rtTA2s-M2 CD200tg mice as recipients. To explore the potential molecular mechanism(s) involved in this allograft tolerance, a commercial microarray kit focusing on detecting altered expression of genes related with T-cell anergy/tolerance was used to investigate the gene expression profile in grafted tissue of mice with transgenic expression of CD200. Expression of genes associated with Foxp3+ regulatory T-populations (Foxp3, CTLA4 and GITR) and type 2 cytokine genes showed increased expression in CD200tg recipients. With particular note in regards to Foxp3+ regulatory T cells, expression of the gene encoding chemokine receptor CCR4, reported to play a key role in attracting Foxp3+ regulatory T cells to grafted tissues and DLNs, was found to be increased in Dox-treated CD200tg recipients, along with genes encoding chemokines CCL22/17, the ligands for CCR4. Immunochemistry staining also showed increased numbers of Foxp3+ cells in both grafted skin tissues and the DLNs of transplant at day 14 post transplantation. Using CCR4-shRNA lentivirus administered to Dox-treated CD200tg recipients to block expression of CCR4, the transgene-induced increased presence of regulatory T cell populations in grafted tissues and DLNs was attenuated, along with loss of enhanced skin graft survival and the histological appearance of graft acceptance (see chapter 4). These data provide support for a model suggesting that altered migration of Treg mediated through a CCR4:CCL17/22 pathway is an important mechanism underlying increased allograft acceptance following CD200tg expression.Ph
Mechanism(s) of Action of the Novel Immunoregulatory Molecule, CD200
No full textBoth CD200 and its receptor(s), CD200R(s), are type I membrane glycoproteins belonging to the immunoglobulin (Ig) supergene family. CD200:CD200R(s) interaction manipulates host immunity in multiple models, including those exploring allograft rejection, autoimmune disease, tumor development, spontaneous fetal loss, infection/inflammation, and virus infection. The studies described in this thesis were focused on investigation possible mechanism(s) involved in CD200-mediated regulation, using transgenic mice over-expressing CD200, and exploring models of skin allograft rejection and LPS-induced abortion in mice.
A Tet-on system was chosen to create CD200tg mice (rtTA CD200tg animal line), in which transgenic expression of CD200 is induced by the presence of doxycycline (Dox-treated mice). Splenocytes from Dox-treated transgenic mice, used as either responder cells or stimulator cells in mixed leukocyte cultures, showed antigen-specific suppressed lymphocyte proliferation and induction of CTL. Although enhanced survival of skin allografts was achieved in Dox-treated transplanted CD200tg mice (BALB/c to BL/6 Tg), all grafts were rejected by 28 days post transplantation (see chapter 2).
A superior “second generation” Tet-on system was used to create a new CD200tg animal referred to as rtTA2s-M2 CD200tg mice. Transgenic overexpression of CD200 in this mouse was stably induced at much lower Dox concentrations, with less (or no) “leaky” expression of the transgene in the absence of Dox. Using these mice in an LPS-induced murine abortion model, transgenic expression of CD200 was found to reduce the LPS-induced abortion rate from ~49% to 6% (see chapter 3).
Long term increased survival of grafted tissues (of both cardiac and skin allografts) was achieved using the rtTA2s-M2 CD200tg mice as recipients. To explore the potential molecular mechanism(s) involved in this allograft tolerance, a commercial microarray kit focusing on detecting altered expression of genes related with T-cell anergy/tolerance was used to investigate the gene expression profile in grafted tissue of mice with transgenic expression of CD200. Expression of genes associated with Foxp3+ regulatory T-populations (Foxp3, CTLA4 and GITR) and type 2 cytokine genes showed increased expression in CD200tg recipients. With particular note in regards to Foxp3+ regulatory T cells, expression of the gene encoding chemokine receptor CCR4, reported to play a key role in attracting Foxp3+ regulatory T cells to grafted tissues and DLNs, was found to be increased in Dox-treated CD200tg recipients, along with genes encoding chemokines CCL22/17, the ligands for CCR4. Immunochemistry staining also showed increased numbers of Foxp3+ cells in both grafted skin tissues and the DLNs of transplant at day 14 post transplantation. Using CCR4-shRNA lentivirus administered to Dox-treated CD200tg recipients to block expression of CCR4, the transgene-induced increased presence of regulatory T cell populations in grafted tissues and DLNs was attenuated, along with loss of enhanced skin graft survival and the histological appearance of graft acceptance (see chapter 4). These data provide support for a model suggesting that altered migration of Treg mediated through a CCR4:CCL17/22 pathway is an important mechanism underlying increased allograft acceptance following CD200tg expression.Ph
Characterization of a Novel Dendritic Cell Population
No full textConventional DC (cDC) arise from circulating immediate precursors (pre-cDC), and are currently thought to be terminally differentiated. Here we show that cDC are capable of generating progeny that lost all characteristic features of cDC and aquired regulatory properties. Sorted bone marrow pre-cDCs were cultured on a stromal monolayer in the presence or absence of granulocyte-macrophage colony stimulating factor (GM-CSF). In the absence of GM-CSF, pre-cDC derived DCs gave rise to a homogeneous population of CD11clow MHClow cells (DC-regs) on day 8-10 of culture. DC-regs failed to up-regulate major histocompatibility complex class II (MHCII) and co-stimulatory molecules in response to DC maturation stimuli, were poor stimulators in T cell proliferation assays and suppressed T cell proliferation in cultures containing immuno-stimulatory DC. Co-transfer of DC-regs with DCs in vivo did not inhibit proliferation of T cells. These findings reveal the potential of DCs to generate a regulatory DC population with immunosuppressive properties.MAS
Checkpoint blockade in solid tumors and B-cell malignancies, with special consideration of the role of CD200
No full textReginald M Gorczynski,1 Fang Zhu2 1Department of Surgical Research, University Health Network, 2Department of Surgical Research, Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada Abstract: In the ontogeny of a normal immune response, a series of checkpoints must be overcome to ensure that unwanted and/or harmful self-directed activation responses are avoided. Many of the molecules now known to be active in this overseeing of the evolving immune activation cascade, contributing inhibitory signals to dampen an overexuberant response, belong to the immunoglobulin supergene family. These include members of the CD28/CTLA-4:B7.1/B7.2 receptor/ligand family, PD-1 and PDL-1, CD200 and CD200R, and the more recently described V-domain immunoglobulin suppressor of T-cell activation and its ligand (VSIG-3/IGSF11). Unfortunately, from the point of view of improving immunotargeting of cancer cells, triggering these checkpoint inhibitory signaling pathways, so necessary to maintain self-tolerance, simultaneously acts to prevent effective tumor immunity. The recent development of reagents, predominantly antibodies, to act as checkpoint blockade agents, has had a dramatic effect on human cancer treatment, with a marked reported success for anti-CTLA-4 and PD-1 in particular in clinical trials. This review provides a general overview of the data now available showing the promise of such treatments to our cancer armamentarium and elaborates in depth on the potential promise of what can be regarded as an underappreciated target molecule for checkpoint blockade in chronic lymphocytic leukemia and solid tumors, CD200. Keywords: checkpoint blockade, immunotherapy, oncology, inhibitory pathways, stimulatory pathways, activated T cell
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