Browsing by Subject "natural killer cells"
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Item Approaches to Cloning and Identification of the Ligand for Natural Cytotoxicity Receptor NKp44(2008-07-01) Horton, Nathan C.; Harlan Jones; Stanley Stevens; Raghu KrishnamoorthyHorton, Nathan C., Approaches to Cloning and Identification of the Ligand for the Natural Cytotoxicity Receptor, NKp44. Masters of Science (Microbiology & Immunology), July 2008, 64 pp., 22 illustrations, 37 titles. Natural Killer (NK) cells represent a specialized lymphoid population that mediate innate immune responses against tumor or virally infected cells. NK cell cytotoxicity is regulated by inhibitory and activating receptors. Activating receptors include the Natural Cytotoxicity Receptors (NCRs), 2B4, and NKG2D. The NCRs play a key role in recognition and killing of tumor cells and include the receptors NKp30, NKp46, and NKp44. The ligands for the NCRs are not yet known. NKp44 is of particular interest because it is only expressed on activated NK cells, and is implicated in increased cytotoxicity and HIV infection. To identify and clone the ligand for NKp44, a recombinant fusion protein containing the extracellular domain of NKp44 was constructed and used to identify a cell line, DB, expressing a ligand for NKp44. A directional complimentary DNA (cDNA) library was constructed from this cell line and screened by mammalian expression cloning, resulting in the isolation of several putative cDNA clones of NKp44 ligands.Item Characterization of a Novel Receptor CS1 in Human Lymphocytes; Studies in Natural Killer Cells and B-Lymphocytes(2005-06-01) Lee, Jae Kyung; Porunelloor Mathew; Ming-Chi Wu; Hriday DasThe purpose of this study was to investigate the roles of CS1 on human lymphocytes. The molecular and functional characterization of CS1 receptor in the natural killer (NK) cells and B lymphocytes was investigated. CS1 (CRACC, novel Ly9) is a novel member of the CD2 family receptor expressed on natural killer (NK), T cells, activated Bcells and dendritic cells. To examine the existence of isoform of CS1, library from NK cells was screened based on wild type of CS1 (CS1-L). A splice variant form of CS1 (CS1-S), which lacks immunoreceptor tyrosine-based switch motifs (ITSMs) in cytoplasmic domain, was identified. To demonstrate the function of CS1 on human NK cells, transfectants that stably express each isoform were generated. CS1-L was able to mediate redirect cytotoxicity of P815 target cells as well as intracellular calcium influx in the presence of monoclonal antibody against CS1 suggesting that CS1-L is an activating receptor. CS1-S showed no effect on the cytolytic function and calcium influx suggesting that CS1-L and CS1-S may differentially regulate human NK cell functions. Although CS1 was also cloned from cDNA library of human B-lymphocytes as well as of NK cells, very little is known regarding its biology on human B-lymphocytes. Here I investigated the expressions and functions of CS1 in human B cells. Human B cells expresses only CS1-L isoform and the levels of CS1 expression are upregulated after activation in vitro. Importantly, monoclonal antibody of CS1 (1G10 mAb) strongly enhances proliferation of both freshly isolated and activated B cells. The enhanced proliferation effects of CS1 were most prominent on B cells activated by anti-CD40 mAbs and/or IL-4. Human cytokine microarray results indicated CS1 enhanced mRNA transcripts of fms-line tyrosine kinase 3 ligand, lymphotoxin A, tumor necrosis factor, and IL-14 which are related with mostly growth promoting activity. These results suggest that autorine cytokines might be the mediators for the function of CS1 on B cell in which it can induce proliferation of activated B cells. This study suggests that CS1 plays important role in human NK cells and B-lymphocytes.Item Characterization of Markers on Pancreatic and Colon Cancer Stem Cells to Enhance Natural Killer Cell Effector Functions(2021-05) Malaer, Joseph D.; Mathew, Porunelloor A.; Mathew, Stephen O.; Berg, Rance E.; Jones, Harlan P.; Prokai, LaszloDue to advancements in technology and medicine, the average human lifespan in the United States has increased to 79 years since the start of the 20th century. This increase in lifespan is also coupled with an increased risk of developing cancer. While cancer detection and initial treatment has increased the survival of patients, it is not a cure and patients wonder how long they will remain in remission. It is estimated that over 90% of cancer related deaths are due to relapse and metastasis. There is currently a growing body of research that indicates cancer stem cells (CSC) are responsible for relapse and metastasis. CSC are a small subpopulation of cells that retain self-renewal and pluripotency. These cells are also described as being quiescent, which may contribute to their chemotherapy resistance. Additionally, CSC are thought to express ligands to aid in immune evasion mechanisms. Natural Killer (NK) cells are lymphocytes of the innate immune system which function to combat infection and cancer. NK cells, unlike T and B cells, do not require prior sensitization. NK cell function is regulated through activating and inhibitory receptors. Recently, Proliferating Cell Nuclear Antigen (PCNA) was identified as an inhibitory ligand for the natural cytotoxicity receptor NKp44. Lectin-like transcript 1 (LLT1) expression is known to facilitate escape of NK cell effector functions in prostate and breast cancer, and now colorectal cancer. In this study we analyzed the expression of molecules on pancreatic and colon cancer cells that could allow escape from NK cell-mediated immune surveillance. The data presented here demonstrates surface PCNA expressing cells as CSC through co-expression of CSC surface markers CD44 and CD133 as well as overexpression of CSC transcription factors NANOG, SOX-2, and Oct-4. Blocking PCNA or NKp44 alters interferon-ℽ secretion by NK cells when cocultured with pancreatic and colon cancer cells. It is further demonstrated that blocking LLT1 or the PCNA-NKp44 interaction led to an increase in specific lysis of tumor cells. This study suggests that cell surface PCNA could function as a biomarker and an immunotherapeutic target for NK cell mediated killing of pancreatic cancer and colon cancer cells.Item Investigation of Proteasome Chymotryptic Activities and Effects on their Inhibition in Rat and Human Natural Killer Cells(2003-04-01) Lu, Min; Goldfarb, Ronald H.; Borejdo, Julian; Easom, RichardLu, Min, Investigation of Proteasome Chymotryptic Activites and Effects of Their Inhibition in Rat and Human Natural Killer Cells. Doctor of Philosophy (Biochemistry and Molecular Biology), April, 2003, 185 pp., 3 tables, 32 illustrations, bibliography, 158 titles. The proteasome is a multicatalytic proteinase complex that is involved in the major extralysosomal pathway responsible for intracellular protein degradation in mammalian cells. This dissertation focuses on investigating proteasome chymotryptic activities and the effects of selective inhibitors of these activities on the function of natural killer (NK) cells. In this dissertation, 20S proteasomes derived from rat RNK16 cells were purified and some of their biochemical and biophysical properties were investigated extensively. The results indicated that RNK16 cell-derived proteasome differ from the proteasome of other origins in many aspects including substrate selectivity, inhibitor specificity, and kinetic regulation, although they may share some common biochemical properties with others. To investigate the effects of proteasomal inhibition on the function of NK cells, several proteasome inhibitors were used including MG115, MG132, clasto-lactacystin-β-lactone, EGCG and LLnL. MG115 and MG 132 were shown to induce apoptosis of RNK16 cells, as evidenced by DNA fragmentation, caspase-3 activation and the appearance of sub-G1 cell populations. Activation of multiple caspases and increased expression of cell surface Fas (CD95) protein were also observed following the treatment of RNK16 cells by these two inhibitors. This dissertation also tested the hypothesis that different cell types could respond differentially to proteasome inhibitors. The effects of several proteasome inhibitors were determined on the purified 20S proteasomal and 26S proteasomal chymotrypsin-like activity in whole cell extracts and intact YT and Jurkat cells, human NK and T cell lines respectively. Following such treatment, caspase-3 activation occurred much earlier in Jurkat cells than YT cells; cell cycle analysis indicated a sub-G1 apoptotic cell population in Jurkat cells and G2/M arrest in YT cells. In addition, accumulation of p27 and IκB-α was detected only in Jurkat cells, but not YT cells. Therefore, proteasome inhibitors appear to act differentially in cell cycle progression and apoptosis signaling pathways between human NK and T cells. These studies indicate that the generation of ideal proteasome inhibitors for the treatment of malignancies could be screened or designed to specifically induce cancer cells to undergo programmed cell death, while having little or no apoptosis-inducing abilities for natural killer cells and other cells of the immune response, thus enhancing the selectivity and specificity of the anti-cancer, apoptosis-inducing capabilities of proteasome inhibitors.Item Molecular Basis for 2B4-CD48 Interactions(2001-08-01) Huynh, Van T.; Mathew, Porunelloor A.; Goldfarb, Ronald; Das, HridayHuynh, Van T., Molecular Basis for 2B4-CD48 Interactions. Master of Science, Molecular Biology and Immunology, August 2001, 93 pp., 3 tables, 19 illustrations, bibliography, 51 titles. Natural killer cells are lymphocytes that play a role against cancer and viral infections. 2B4 is a membrane glycoprotein expressed on natural killer cells. In the present study we characterized 2B4 from mice strains BALB/c, 129/svj and A.CA. Nucleotide and peptide analysis revealed that polymorphyic residues in 2B4 are located in the variable domain. My second project was to determine the amino acids involved in the binding between 2B4 and CD48. Twelve mutations were made in human 2B4 to disrupt their interaction. In the last part of the study, an attempt has been made to elucidate the role of tyrosine and threonine amino acids found in the novel tyrosine motifs (TxYxxI/V) that reside in the cytoplasmic domain.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 The Functional Role of Human 2B4 (CD244) Isoforms in Natural Killer Cells(2007-05-01) Rao, Krithi K.; Porunelloor Mathew; Rance Berg; Harlan JonesRao, Krithi K., Functional role of human 2B4 (CD244) isoforms in natural killer cells. Master of Science (Immunology), July, 2007, 66 pp., 15 illustrations, bibliography. Natural killer (NK) cells are a subpopulation of lymphoctyes that play an important role against tumor metastasis and various viral and bacterial infections. NK cell functions are controlled by a balance between positive and negative signals through various receptors. We have identified, cloned, and characterized the 2B4 (CD244) receptor in mice and human. 2B4 is involved in killing cancer cells and virus-infected cells by NK cells. 2B4 is involved in killing cancer cells and virus-infected cells by NK cells. 2B4 is a counter-receptor for CD48 and recent findings show that 2B4-CD48 interactions plan an important role in NK, T and B cell functions. In humans, two isoforms of 2B4, h2B4-A and h2B4-B, are expressed that differ in the extracellular domain. In the present investigation, we have studied the functions of h2B4-A and h2B4-B. Our data demonstrate that these two isoforms differ in their binding affinity for CD48, resulting in differential cytolytic function as well as cytokine production by NK cells. Thus, differential expression of 2B4 isoforms by NK cells may regulate immune responses mediated through 2B4-CD48 interactions.