Browsing by Subject "Immunology"
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Item AGING-INDUCED INCREASED SUSCEPTIBILITY TO AUTOIMMUNITY IS DUE TO COMPROMISED NEGATIVE SELECTION IN THE THYMUS RATHER THAN DEFECTS IN REGULATORY T CELLS(2013-04-12) Coder, BrandonPurpose: Immunotolerance generates protection against autoimmunity by deleting self reactive T cells in the thymus through negative selection as well as the generation of natural regulatory T cells (nTregs) that will help suppress autoimmunity in the periphery. Natural aging is associated with a progressive loss of FoxN1 and thymic atrophy, and is also believed to be associated with increased autoimmunity and an increase in suppressive FoxP3+ regulatory T cells (Tregs). Deletion of self-reactive T cells requires self-antigen presentation by medullary thymic epithelial cells (mTECs). We set out to determine if thymic aging, characterized by the progressive loss of FoxN1 and mTEC disruption, alters immunotolerance by influencing negative selection or impacting the generation of suppressive Treg cells. Methods: For thymus population experiments, we induce thymic atrophy in our FoxN1 conditional knockout mice using tamoxifen injections. We then determine expression of CD4+, CD8+, and CD4+8+ double positive, and CD25+FoxP3+ regulatory thymocytes using flow cytometry. The peripheral Treg data is assessed by adoptive transfer of total spleenocytes from young and aged wild type mice into young Rag2-/- mice. Cell populations are determined using flow cytometry. Results: We found that the loss of FoxN1 induced thymic atrophy is associated with an impairment of negative selection, where CD4+ and CD8+ are increased in the thymus while CD4+CD8+ double positive thymocytes are decreased. This indicates that the age atrophied thymus is not able to delete additional single positive thymocytes, and these may be self-reactive thymocytes. Additionally, we found that nTregs in the aged and atrophied thymus are increased in proportion and their suppressor function remains intact. Furthermore, we found that when we transfer aged spleenocytes, in which there are increased Treg and decreased pro-apoptotic Bim protein, and young spleenocytes separately into young Rag2 knockout mice, that the young periphery is able to restore both Bim levels and Treg levels to that of the young mice. Conclusions: We conclude that loss of FoxN1 disrupts thymic mTEC structure and impairs negative selection, which may lead to an increase in self-reactive T cells. However, thymic atrophy does not compromise peripheral Tregs. The function and number of peripheral Tregs is dependent on the micro-environment in which they stay.Item EXTRACELLULAR PROLIFERATING CELL NUCLEAR ANTIGEN IS A NOVEL MARKER FOR CANCER STEM CELLS AND FACILITATES EVASION OF NK CELL EFFECTOR FUNCTION(2013-04-12) Horton, NathanPurpose: Natural Killer (NK) cells are a specialized population of lymphocytes of the innate immune system which provide vital first line defense against infections and cancer. NK cell function is strictly regulated by inhibitory and activating receptors binding corresponding ligands on the surface of target cells. NKp44, originally discovered as an activating NK cell receptor, was recently found to elicit inhibitory effects on NK cell effector function through recognition of cell surface Proliferation Cell Nuclear Antigen (PCNA), which is typically only found in the cell nucleus for DNA replication and repair. Other reports have pointed to potential associations between NKp44 and Human Leukocyte Antigen (HLA) I molecules. Methods: We have identified novel interactions between HLA I and PCNA on the surface of human tumor cells by confocal microscopy and immunoprecipitation. We have also confirmed the inhibitory nature of NKp44 recognition of PCNA in association with HLA I through a standard Chromium release assay. Results: We show PCNA on the cell surface in novel association with HLA I is a natural process which does not require provocation and occurs with endogenous PCNA. The association of HLA I and PCNA forms the inhibitory ligand for NKp44, resulting in inhibition of NK effector function. Furthermore, extracellular PCNA was only found on tumor cells exhibiting a cancer stem cell phenotype. Conclusions: Thus extracellular PCNA may serve as a novel marker for cancer stem cells and provide a mechanism by which these cells evade NK cell effector functions. As a rare population of cancer cells potentially responsible for relapse after treatment, metastasis, and overall tumor growth, cancer stem cells are difficult to clinically detect in circulation and are often impervious to chemotherapy and radiation treatments. Further studies will determine the efficacy of using extracellular PCNA as a cancer stem cell marker for better detection and therapeutic targeting of these rare, yet dangerous cancer cells.Item NOVEL USE OF PROLIFERATING CELL NUCLEAR ANTIGEN AS A BIOMARKER OF METASTATIC CANCER(2014-03) Horton, Nathan; Mathew, Porunelloor A.Novel biomarkers to identify metastatic tumor cells are needed to better identify these cells and to appropriately choose therapeutic measures. Cancer stem cells are believed to be responsible for metastasis and relapse after therapy. We have identified novel expression of Proliferating Cell Nuclear Antigen on the cell surface of tumors and characterized these cells as potential cancer stem cells. This research may facilitate the generation of novel immune based cancer therapies to specifically identify and target metastatic tumor cells. Purpose (a): Primary tumors account for 10% of cancer related deaths. Thus, identifying novel biomarkers on tumor cells which resist treatment or potentially become metastatic is vital. Proliferating Cell Nuclear Antigen (PCNA) has traditionally been used as a biomarker to identify and grade tumor biopsies based on PCNA's involvement in DNA replication. Typically located intracellularly, we have recently identified PCNA at the cell surface. When recognized by the Natural Killer (NK) cell receptor, NKp44, PCNA inhibits NK cell effector functions, allowing tumor cells to escape immunosurveillance. We have characterized tumor cells expressing cell surface PCNA to evaluate the use of cell surface PCNA as a potential marker of cancer stem cells, believed to be responsible for relapse and metastasis. Methods (b): We analyzed extracellular PCNA expressing tumor cells for expression of vimentin by confocal microscopy and expression of CD44 and CD24 by flow cytometry, which mark cancer stem cells. We also analyzed these cells for expression of genes which can induce formation of cancer stem cells or maintain stem cell characteristics by real time PCR. Finally, since stem cells are often quiescent, we analyzed cell cycle progression of these cells using propidium iodide and flow cytometry analysis. Results (c): Expression of vimentin is exclusive to cells expressing extracellular PCNA. These cells also express intermediate levels of CD44, which marks metastatic cells in vivo, and differentially express genetic markers of cancer stem cells. Populations of tumor cells expressing PCNA at the cell surface were enriched for cells in the G2/M phase of the cell cycle. Conclusions (d): Extracellular PCNA may be a marker for metastatic cancer stem cells based on intermediate expression of CD44, concomitant expression of vimentin, and expression of genetic markers. Alternatively, extracellular PCNA marks cells in the G2/M phase. Further studies in mouse models will be needed to confirm extracellular PCNA as a marker for cancer stem cells.