Browsing by Subject "NK cells"
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Item ALTERED EXPRESSION OF IMMUNE RECEPTORS 2B4, CS1 AND LLT1 IN CHILDREN WITH ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)(2013-04-12) Tan, CharissePurpose: Acute lymphoblastic leukemia is the most common type of leukemia found in children with peak prevalence between the ages of 2 and 5. In ALL, there is uncontrolled, exaggerated growth and accumulation of lymphoid progenitor cells, which fail to properly differentiate and function as normal lymphocytes. Despite current treatment protocols that have been successful in the vast majority of ALL patients, serious acute and late complications are frequent and resistance to chemotherapy often develops. Natural Killer (NK) cells, which are components of the lymphocyte population, can recognize and act on target cells under the control of their cell surface receptors. Binding of these receptors to specific ligands on the target cell results in signaling which either activates or inhibits NK cell effector functions. We have previously identified cell surface receptors 2B4, CS1 and LLT1 playing a major role in NK cell activation. Previous studies on these receptors implicate that these receptors may play a role in cancers, however their significance and role in childhood ALL have not been evaluated. We hypothesize that altered expression of these immune receptors play a role in acute lymphoblastic leukemia in children. Methods: ALL subjects and healthy subjects were enrolled at Cook Children's Hospital and UNT Health Science Center, Fort Worth, TX with informed consent/assent according to IRB approval. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood of twelve ALL subjects and three healthy subjects. NK92-MI cell line was used as a positive control. Total RNA was extracted and reverse transcribed (RT-PCR) into complimentary DNA (cDNA). PCR was performed using primers specific to 2B4, CS1, LLT1, and expression was normalized to β-actin. Results: ALL subjects showed altered expression of 2B4 in PBMC as compared to healthy subjects. There was an overall decrease in the expression of CS1 receptor in ALL subjects as compared to healthy subjects. Interestingly, both ALL and healthy subjects showed expression of different isoforms of the LLT1 receptor with variations in their expression level. Conclusions: Results implicate that there are alterations in the expression of immune receptors that may mediate the immune dysregulation in ALL subjects. Expression and functional analysis of these receptors in a larger population of ALL subjects will provide vital knowledge furthering our understanding of the etiology of ALL, progression, and developing potential immunotherapies.Item Evaluation of NK Cell – Astrocyte Interactions: Potential Role in HIV-Associated Neurocognitive Disorders and HIV- Associated Dementia(2015-05-01) Bowen, Kelly E.; Mathew, Porunelloor A.; Mathew, Stephen O.; Hodge, Lisa M.NK cells play important roles in immunity against pathogens and cancer. NK cell functions are regulated by inhibitory and activating receptors binding corresponding ligands on the surface of target cells. During pathological conditions, NK cells were shown to be recruited to the CNS and could impact CNS physiology by killing glial cells and by secreting IFN-g. Astrocytes are intimately involved in immunological and inflammatory events occurring in the CNS and reactive astrogliosis is a key feature in HIV-associated neurocognitive disorders (HAND). There is little data on NK cell-astrocyte interactions and ligands expressed on astrocytes that could impact NK cell function. This study aimed to identify NK-associated ligands expressed by human astrocytes that confer this NK-directed cytotoxicity of astrocytes and assay the cytotoxicity differences in presence and absence of HIV 3S peptide. Using a fusion protein consisting of the extracellular domain of NKp44 fused to Fc portion of human IgG, we determined the expression of a novel ligand for NKp44 (NKp44L) on astrocytes. Incubation of astrocytes with 3S peptide downregulated NKp44L expression on astrocytes implicating protection from NK mediated killing. Thus, our study demonstrated that NKp44 has a protective effect on astrocytes from NK cell mediated killing during HIV infection. Astrocytes could also secrete cytokines that affect the expression of NK receptors on NK cells. We evaluated the expression of receptors on NK cells after co-culture with astrocytes. CD38 expression was increased on primary NK cells after incubation with astrocytes. CD38 is expressed on both NK cells and astrocytes and has an important implication in HIV-1 infection. Blocking CD38 signaling in our studies decreased astrocyte lysis, suggesting CD38 signaling has important implications in NK-astrocyte interactions. Future studies providing novel insights into the role of NK cells in the pathogenesis of HAND and other brain disorders might result in the development of NK cell based therapies for brain pathologies.Item Molecular Cloning and Regulation of Expression of an NK Cell Receptor(2001-07-01) Medina, Miguel Angel; Porunelloor Mathew; Rafael Alvarez-Gonzales; Neeraj AgarwalNatural killer (NK) cells are large granular lymphocytes derived from bone marrow. They form the first line of defense against virally infected and tumor cells. Unlike B and T cells, they are not MHC restricted therefore do not require prior antigen stimulation (1-4). NK cell functions include producing various cytokines such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and granular-macrophage colony stimulating factor (GM-CSF) and cytotoxicity (5,6). A number of cell surface molecules have been identified, cloned and characterized that modulate NK cell recognition and activation by target cells (1). Most of these molecules are also expressed on other leukocytes. NK cell function is regulated by the balance of the positive and negative signaling through these receptors (3, 7-10). In the past attention has primarily focused on major histocompatibility complex (MHC) recognizing receptors that are mostly inhibitory (11). It is through these inhibitory receptors that levels of MHC molecules and associated peptides are monitored. Cells that have lost the expression of MHC class I molecules or have altered peptides-class I complexes are not able to transmit an inhibitory signal to NK cells and are consequently killed. Members of the CD2 subset of receptors play a major role in lymphocyte functions and do not recognize MHC molecules. The signaling lymphocyte activation molecule, SLAM (CD150), a member of the CD2 subset, is expressed on T cells and B cells. SLAM regulates T cell activation and production of immunoglobulins by B cells (12,13). 2B4 is a member of the CD2 subset and is expressed on NK cells as well as other leukocytes (14, 15). 2 B4 is a surface molecule implicated in the activation of NK cell-medicated cytotoxicity (15-17). Human 2B4 is a 60-70 kDA glycoprotein surface molecule found on all NK cells and a small subset of T cells that exhibit NK-like activity. CD48 has been identified as the high affinity ligand for 2B4 and implicates a broader role for 2B4 in immune regulation (18, 19). Recent reports have demonstrated the importance 2B4 and the functional role 2B4 plays in immune regulation. In X-linked lymporoliferative (XLP) disease NK cells can not be activated through surface 2B4 (20-23). The molecular adaptor protein, SLAM-associated protein or SH2 domain containing adaptor molecule (SAP/SH2D1A) is associates with cytoplasmic tail of 2B4 or SLAM (24, 25). Defective signaling via 2B4 and SLAM may contribute to the pathogenesis of X-linked lymphoproliferative disease due to mutations in SAP. The cytoplasmic domain of 2B4 contains four novel tyrosine motifs (TxYxxV/I) (14, 15). SLAM, a close relative of 2B4, also contains these novel tyrosine motifs. The signaling mechanism for 2B4 remains unclear. Along with other members of the CD2 subset 2B4 also localizes to chromosome 1. The genes that encode the CD2 family of receptors are locatedon human chromosome at 1q21-24 (24, 26-30). The murine genes for 2B4, CD48, Ly49, Ly108, and CD84 are located on the syntenic region of the long arm of the chromosome 1 (30-33). The exon arrangement for 2B4 is consistent with other CD2 subset members and consists of an exon per domain for the leader sequence, V-like domain, C2-like domain, and the transmembrane domains (27, 34-37). Differential exon usage leads to splice variants of the receptors, which complicates understanding the functional relevance between the cytoplasmic domains between receptors. Both murine 2B4 and SLAM demonstrates splice variants that alter the number of novel tyrosine motifs within the cytoplasmic domains (14, 34, 38). The murine 2B4 gene consists of 9 exons with one exon dedicated to each leader sequence, V-like, C2-like, and transmembrane domains. The total gene size is approximately 27 kilobases with the first intron consisting of 16 kilobases. Variable exon usage gives rise to two isoforms of 2B4, 2B4-L and 2B4-S, in the mouse (38). Four exons encode the 2B4-L cytoplasmic domain, giving rise to four tyrosine motifs. 2B4-S is identical to the 5’end of 2B4-L, differing only at the 3’ end in a portion of the cytoplasmic domain and the 3’untranslated sequence. 2B4-S is the product of the same first five exons in 2B4-L with the usage of a novel exon at the C-terminal. Although splice variants exists there Is no direct biochemical evidence to support their expression. In vitro analysis of the m2B4 variants suggest potential signaling differences. Murine 2B4 variants and mutants were transfected into a rat NK cell line, RNK-16. Interestingly, the two forms of 2B4 had opposing functions (39). Murine 2B4 is expressed on all NK cells, a subset of T cells, dendritic epidermal T cells, and monocytes (40). Expression levels of 2B4 can be elevated by incubation with interleukin-2 (IL-2). Engagement of 2B4 can be elevated by incubation with interleukin-2 (IL-2). Engagement of 2B4 with anti-2B4 monoclonal antibody (mAb) causes secretion of interferon-γ, increased 2B4 expression, and elevated cytotoxicity (41). Characterization of how 2B4 and its related receptors are expressed is critical to the understanding not only the receptors’ biology but also NK cell biology. My first project will focus on mastering the techniques involved in the isolation and characterization of genes. Previously two genomic clones were isolated from 129 Sv/J mouse liver, 531 and 532. The first clone, 531, has been fully characterized and revealed to be 2B4. 532 has been partially characterized and revealed to the related form of mouse 2B4. In order to determine the function of 532 on mouse NK cells, 532 cDNA has to be isolated. I attempted to isolate 532 cDNA through PCR using previously isolated clones from the BALB/c cDNA library. My next aim was to isolate 532 genomic DNA for automated sequencing. I used this data to design primers specific for 532 and isolate the 532 cDNA through RT-PCR. 532 will be the topic discussed in chapter 2 and chapter 3 will discuss the 2B4 activated sequencing. I used this data to design primers specific for 532 and isolate the 532 cDNA through RT-PCR. 532 will be the topic discussed in chapter 2 and chapter 3 will discuss the 2B4 activated response molecule. The final portion of my thesis will focus on the isolation of the 2B4 activated response molecule (2ARM). Human peripheral blood NK cells were isolated incubated with interleukin-2 or C1.7. C1.7 is a monoclonal antibody that specifically recognizes human 2B4. RNA was extracted from these NK cells at various time points and used for RT-PCR to monitor the expression levels of 2B4. Aside from the expression of human 2B4, the expression of a 160 base pair transcript was also detected. Sequencing analysis revealed this transcript to be novel. I screened a human NK cDNA library constructed by Dr. J. Houchins (R & D System, Minneapolis, MN) using this 160 base pair transcript as a probe. Upon isolation of 2ARM cDNA, functional analysis can be performed to determine its role on human NK cells.Item Overexpression of LLT1 (OCIL, CLEC2D) on prostate cancer cells inhibits NK cell-mediated killing through LLT1-NKRP1A (CD161) interaction(Impact Journals, LLC, 2016-09-08) Mathew, Stephen O.; Chaudhary, Pankaj; Powers, Sheila B.; Vishwanatha, Jamboor K.; Mathew, Porunelloor A.Prostate cancer is the most common type of cancer diagnosed and the second leading cause of cancer-related death in American men. Natural Killer (NK) cells are the first line of defense against cancer and infections. NK cell function is regulated by a delicate balance between signals received through activating and inhibitory receptors. Previously, we identified Lectin-like transcript-1 (LLT1/OCIL/CLEC2D) as a counter-receptor for the NK cell inhibitory receptor NKRP1A (CD161). Interaction of LLT1 expressed on target cells with NKRP1A inhibits NK cell activation. In this study, we have found that LLT1 was overexpressed on prostate cancer cell lines (DU145, LNCaP, 22Rv1 and PC3) and in primary prostate cancer tissues both at the mRNA and protein level. We further showed that LLT1 is retained intracellularly in normal prostate cells with minimal cell surface expression. Blocking LLT1 interaction with NKRP1A by anti-LLT1 mAb on prostate cancer cells increased the NK-mediated cytotoxicity of prostate cancer cells. The results indicate that prostate cancer cells may evade immune attack by NK cells by expressing LLT1 to inhibit NK cell-mediated cytolytic activity through LLT1-NKRP1A interaction. Blocking LLT1-NKRP1A interaction will make prostate cancer cells susceptible to killing by NK cells and therefore may be a new therapeutic option for treatment of prostate cancer.Item The Role of LLT1 (CLEC2D, OCIL) in Ewing Sarcoma(2021-12) Buller, Casey W.; Mathew, Stephen O.; Mathew, Porunelloor A.; Jones, Harlan P.; Basha, Riyaz; Tovar-Vidales, TaraEwing Sarcoma (EWS) is a pediatric bone cancer that is characterized by a chromosomal translocation giving rise to a neomorphic gene fusion. Although treatment of EWS has a 5-year incident free survival rate of 66%, treatment has plateaued since the 1980's. Natural killer (NK) cells are an important innate immune cell due to their ability to recognize and lyse virally infected and cancerous cells. Unfortunately, cancerous cells often employ strategies such as the downregulation of major histocompatibility complex (MHC) I, or upregulation of inhibitory ligands enabling the escape of T-cells and NK cell mediated killing. LLT1 is an inhibitory ligand our lab had previously shown that it is expressed on TNBC and prostate cancer cell lines. The expression of LLT1 and its function in Ewing Sarcoma (EWS) cell lines has yet to be performed. Our hypothesis is that LLT1 is increased in EWS cell lines TC-32 and CHLA-258 when treated with chemotherapeutic drugs vincristine and etoposide. Our results show that LLT1 is expressed on EWS cell lines and inhibition of LLT1 increases NK cell cytotoxicity. These results indicate that LLT1 is a potential immunotherapy target that needs to be further evaluated in an in vivo model.Item The Urokinase Plasminogen Activator System in NK Cells: Its Role in Invasion And Its Regulation by IL-2(2002-05-01) Al-Atrash, Gheath; Goldfarb, Ronald H.; Kitson, Richard P.; Mazar, Andrew P.Al-Atrash, Gheath, The Urokinase Plasminogen Activator System in NK Cells: Its Role in Invasion and its Regulation by IL-2. Doctor of Philosophy (Biomedical Sciences), May 2002; 202 pp.; 2 tables; 20 figures; bibliography, 143 titles. Adoptively transferred natural killer (NK) cells can infiltrate tumors and directly kill malignantly transformed cells without prior sensitization. This makes NK cells ideal for cancer immunotherapy, not only for their tumoricidal capacities, but also as drug carriers. This dissertation investigates the role of NK cell urokinase plasminogen activator (uPA) system in NK cell invasion through extracellular matrices (ECMs), its cooperation with matrix metalloproteinases (MMPs), the mechanism by which interleukin-2 (IL-2) upregulates NK cell uPA/uPAR, and the in vivo antitumor therapeutic potential of NK cells as drug delivery vehicles. uPA and its receptor uPAR were detected in human and rat NK cells using RT-PCR, casein plasminogen zymography, western blots, and fluorescence microscopy. In vitro invasion assays showed a role for the uPA system in NK cell invasion, alone and in cooperation with MMPs: this was achieved by using selective plasmin inhibitors in combination with selective MMP inhibitors in ECM/Matrigel invasion assays. uPA’s regulation by the ECM proteins collagen type IV, laminin, and fibronectin was investigated and results show a downregulation of NK cell uPA mRNA by these proteins. IL-2, however, a potent NK cell stimulator, increases both uPA and uPAR, coinciding with an increase in NK cell invasion. This IL-2 upregulation has transcriptional and posttranscriptional components, the latter mediated by uPA and uPAR destabilizing mRNA binding proteins. The use of NK cells as drug delivery vehicles was illustrated by in vivo studies which demonstrated that NK cells linked to doxorubicin were more efficacious in prolonging the survival of tumor bearing mice than either treatment alone. The work presented in this dissertation has substantial impact on the field of adoptive immunotherapy for cancer treatment. Engineering NK cells to transiently express high amounts of uPA and/or uPAR may increase the invasive capacities of NK cells, resulting in greater infiltration of tumors by ex-vivo activated NK cells. Moreover, this enhanced infiltration may allow a greater delivery of anticancer drugs to established metastatic tumors. This can potentially lead to more efficacious and possibly curative NK mediated adoptive immunotherapy, thereby constituting a novel means to overcome current limitations to NK cell-mediated adoptive therapy of cancer metastases.