T cells are type of white blood cell that take part in the body's immune defence against virus cells, foreign cell, and cancer cell elimination. T-cells undergo development in the thymus and migrate to the peripheral lymphoid organs, where foreign antigens will activate them. Over the past two decades numerous studies found a correlation between the regulation of HDAC and several forms of cancer, more recent studies found the inhibition of HDAC to be inducers of growth arrest, differentiation, and/or apoptotic cell death of transformed cells in vitro and in vivo. HDAC inhibitors are now being used as potential cancer treatment. Hydroxamic acid-based hybrid polar compounds are one class of this new cancer therapeutic, chosen for their ability to act selectively, inhibiting tumour cell growth at levels that have little or no toxicity for normal cells. With HDAC inhibiting treatments being targeted at T-cell lymphomas it is the aim of this project to identify the mechanisms by which these drugs induce there effect at a molecular level on T-cell production.
Through the use of conditional knockout mice, genetically modified to lack the expression of HDAC1, HDAC2 or HDAC1/2, the assessment of the role of Histone Deacetylases (HDAC) in T-cell development and function in vitro is being assessed. Following analysis of the spleen, lymph nodes, and thymus for the presence of CD4+ and CD8+ T-cells using various mouse genotypes, the affect of the absence of HDAC in CD4/CD8 populations is being assessed. Our working hypothesis is that inhibition of HDAC 2 activity leads to a substituted production by HDAC1; in contrast, conditional mice containing a deletion of the HDAC1 lack substituted production by HDAC2.
Over the next three months, once a direct link has been established between HDAC1/2 and CD4 or CD8 production levels, we will attempt to characterise these subpopulations, determining whether the presenting populations are that of mature or immature status via death assays.
Introduction:
The lymphoid system consists of several lymphoid organs including the spleen, lymph nodes, bone marrow, thymus and mucosa-associated lymphoid tissue (MALT). These concentration, interaction and deployment of lymphocytes. There are four main classes of lymphocytes involved in the monitoring and regulation of antigen (Ag) intrusions: lymphocytes, natural killer (NK) cells, macrophages and dendric cells. For the purpose of this study, we will focus on lymphocytes, and more specifically thymus derived lymphocytes (T-cells).
Lymphocytes are one of the five kinds of white blood cells or leukocytes that consist of two main types of immune cells, namely the bursa of Fabricius cells (B-cells) and T-cells. B-cells are derived from bone marrow stem cells and develop within the bone marrow. Final maturation occurs in the spleen whereby mature b-cells express surface immunoglobulin (Ig) that act as receptors for specific antigens, where they are primarily involved in the production of antibodies. T-cells also derive from the bone marrow, but unlike B-cells, these hematopoietic stem cells migrate to the richly innervated thymus, a gland with well-defined cortical and medullary regions, where they mature.
T-cells are required for full immunity expression and possess two forms of T-cell receptors (TCR): Alpha-Beta (αβ) whose TCR is a heterodimer of an alpha chain with a beta chain, and Delta-gamma (γδ) whose TCR is also a heterodimer of a gamma chain paired with a delta chain. Both classes of TCR do not occur autonomously
Headings to look into for intro:
T-cell subset differentiation
T-lymphocyte progenitors develop from multipotent hemopoietic foetal liver-derived stem cells in utero (Jotereau et al 1987) and bone marrow progenitors in adult life (Scollay et al 1986; Donskoy and Goldschneider 1992) where they migrate via the blood stream for maturation to the thymus and begin to express an array of surface glycoproteins. The prothymocyte enters the thymic cortex consisting of specialized stromal cells; in embryonic mice Vγ3-bearing cells form via TCR-gene rearrangement that mature into Thy-1 + dendritic epidermal γδ cells found in the skin of adults (Havran and Allison 1990). During adult lymphoid development of early T and B lineage cells the enzyme terminal deoxynucleotidyl transferase (TdT), an enzyme that catalyzes the repetitive addition of dNTPs to the 3´-OH end of a DNA fragment, becomes expressed during TCR-gene rearrangement. TdT increases TCR and the immunoglobulin repertoire diversity during antigen-receptor gene rearrangement by insertion of nucleotides at the template-independent (N region), D and J junction sites (Alt and Baltimore 1982.; Komori el al 1993). Adult mice lacking TdT due to a gene mutation were found to possess an immature lymphocyte repertoire with only a small number of N nucleotides (Gilfillan et al 1993). IL-2 and IL-2R become expressed leading to autocrine proliferation of T-lymphocytes.
T-lymphocytes begin their cell cycle as Th0 type cells (Biedermann et al 2004). Rearrangement of the TCR-genes (αβ/ γδ) takes place where T-lymphocytes progeny progress to either
Cytotoxic T-lymphocytes that inflict direct damage to target cells as a result of various mechanisms such as CD95 or the perforin/granzyme system and also the production of effector cytokines such as TNF-a or IFN-γ (Russell and Ley 2002), characterised by the surface expression of CD8,
Helper T-lymphocytes, characterised by CD4 expression,
CD4+ cells recognize antigens presented in the context of Class II major histocompatibility complex (MHC), while CD8 positive cells recognize antigen presented in the context of Class I MHC.
At this stage, the pre-T-lymphocytes are CD3+CD4-CD8- or "double-negative" cells. Double-negative cells that productively rearrange gamma and delta chain gene segments develop into CD3+CD4-CD8- gamma/delta T-lymphocytes that become exported to the periphery in small numbers. Successful rearrangement of a set of TCR genes suppresses further rearrangement of TCR genes on the sister chromatid (allelic exclusion), thus each cell only expresses TCR with a single specificity. The majority of double-negative cells will go on to rearrange α and β-chain gene segments. The β-chain genes rearrange and are expressed first with a pre-T α-chain to form the pre-TCR. Once the pre-TCR recognizes an intrathymic ligand, a signal is generated and transmitted through CD3 which: [1] Halts further beta chain gene rearrangement (allelic exclusion) [2] Enhances alpha chain gene rearrangement [3] Causes CD4 and CD8 to be expressed. These immature double-positive T-lymphocytes express both CD4+ and CD8+ and later mature into single-positive T-lymphocytes, allowing them to become categorized based upon the loss of a cell surface co-receptors expression of either CD4+ CD8- or CD4- CD8+ .
In 1961, Miller discovered the importance of the thymus in T-lymphocyte development when carrying out studies of neonatal thymectomized mice (Miller J.F.A.P 1961). In 1986, Mosmann et al. published their findings of two identified subsets of activated CD4 positive T-lymphocytes, Th1 and Th2 cells, which differed from each other amongst mouse CD4+ T-lymphocytes clones, (Mosmann et al. 1986; Cherwinski et al. 1987), and later where identified amongst human T-lymphocytes (Del Prete et al. 1991) by their pattern in cytokine antigen-induced production of cytokines and there effector functions.
Th1 T-lymphocyte response:
Th1-immune responses involve
Th2 T-lymphocyte response:
T-cells recognize their specific antigens through the TCR
Significance of T-cell selection to autoimmunity
Histones:
Histones can be defined as small, highly basic, acid soluble proteins (Bloch, D. P. 1963) that associate with nuclear DNA. In eukaryotes, DNA is wrapped around a histone octamer comprising of two copies of each nucleosomal core histones (H2A, H2B, H3, and H4), providing a nuclear scaffold of repeated units of chromatin called the nucleosome. The term epigenetics refers to the change(s) in genetic expression and cellular phenotype without alterations in the DNA sequence itself. Two enzymes involved in determining the state of acetylation of histones are histone acetyl-transferases and histone deacetylases, of which both play a major role in the regulation of gene expression. (Grunstein, 1997). Many studies over almost two decades have reference to an altered HAT or HDAC activity being present in several cancers (Muraoka et al. 1996; He et al. 1998; Lin et al.1998; Timmerman et al. 2001).
Histone Acetyl-transferase (HAT):
Histone acetylation has been found over the years to play an integral role in transcriptional regulation, DNA replication and repair. Acetylation also appears to play a critical role in influencing histone interactions with specific non-histone regulatory proteins (Brownell and Allis, 1996). A group of enzymes collectively known as histone acetyltransferase (HATs) catalyses the conversion of positively charged primary ε-amines to uncharged secondary amines in which acetate from the substrate acetyl-CoA is covalently added to specific lysine residues of histone (Pazin and Kadonaga 1997). There are two types of HATs (A and B), categorised based on their subcellular localisation and substrate specificity (Brownell and Allis, 1996)
A-type HATs (GCN5, PCAF, MORF, MOZ, TIP60, etc) carry out nuclear histone acetylation, related to transcription. (Brownell and Allis, 1996). Cytoplasmic B-type HATs catalyze acetylation of newly synthesized H4, transported from the cytoplasm to newly replicated DNA at the nucleus. (Ruiz-Carrillo et al. 1975; Allis et al. 1985)
Histone Deacetylase:
HATs and histone deacetylases (HDACs) control the steady-state levels of histone acetylation, where HATs catalyse histone acetylation and HDACs perform the reverse action. HDACs control gene transcription by regulating acetylation of DNA sequence-specific transcription factors (Gu and Roeder, 1997; Wilson et al., 2006). Through these mechanisms, HDACs are emerging as critical regulators of gene expression with studies showing several HDAC inhibitors (HDACi) having successful results in the treatment of malignant cells where HDACi shown to revert the malignant phenotype In an article by Minucci et al 2001
The histone switch: HAT and HDAC activities regulate the acetylation status of chromatin. Acetylation establishes a structure that permits ATP-dependent chromatin remodelling factors to open promoters. Acetylated histone tails are shown as purple circles.
http://www.nature.com/embor/journal/v4/n10/full/embor941.html#B16
Class 1 Deacetylases:
HDAC1 was the first of the histone deacetylase family to be identified in mammals. It is a metalloenzyme
*Histone acetylation is a particularly important modification of histone amino-termini, because, in general, increased levels of histone acetylation (hyperacetylation) are associated with transcriptionally permissive chromatin, whereas decreased levels of acetylation (hypoacetylation) are associated with repression of gene expression (Marks et al., 2003.))
Class 1 Deacetylases:
Histone Deacetylase Inhibitors:
Over-expression of various HDACs in many cancer types have lead to HDAC inhibitors being extensively investigated as potential anticancer agents in recent years. In a study carried out by Van Lint et al. where two different forms of HDACi were investigated for their effect on histone hyperacetylation on gene expression. It was found that these forms of inhibitors are highly selective, exerting their affect on transcription on <2% of expressed genes in cultured cells. Although these drugs are becoming more rapidly approved for the treatment in cancer, such as oral vorinostat (Zolinza), used to treat cutaneous manifestations in patients with cutaneous T-cell lymphoma (CTCL), a form of non-Hodgkin's lymphoma. CTCL is a cancer of the T cells that affects the skin. The concerning the primary mechanism by which these agents trigger cell death specifically in tumour cells
HDAC inhibitors as monotherapy appear to have minimal clinical activity, and their biggest role will likely be in combination with other therapeutic agents.
Non-histone deacetylases:
Non-histone proteins whose expression may be altered by HDAC inhibitors include heat shock protein 90 (hsp90), oestrogen receptor (ER)-α, androgen receptor (AR), HER2, p53, and vascular endothelial growth factor (VEGF).
The study of HDAC inhibitors is still in its infancy. While this class of agents holds great promise as anticancer therapy, we have yet to learn how best to administer these drugs. In this era of personalized medicine, we strive to individualize therapy so that maximal benefit may be achieved and unnecessary toxicity minimized. Our ability to do so depends on furthering our understanding of the various mechanisms by which HDAC inhibitors exert their effects, elucidating the optimal sequence and schedule of administration, and identifying individuals who are most likely to benefit from this particular therapy.
**Lagger, G. et al. ( 2002) Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression. EMBO J., 21, 2672-2681.
Activation of cell lines (Thy and Spleen/LN)
P/Ca2+
Headings to look into for methods:
Mice:
Production of T-cell Lines
Re-stimulation of T-cell Lines
Proliferation Assay
Analysis of the T-cell development using Flow Cytometry
cDNA Synthesis
Reverse-Transcriptase Polymerase Chain Reactions (RT PCR)
Gel Electrophoresis
Section 2: Material & Methods
Equipment:
Autoclave
Automatic Pipette (Pipetteman)
Balance (Mettler - AEZOO)
Centrifuge (Jouan - Model 412)
Falcon Tubes - 50ml and 15ml (Corning)
Freezer (Forma scientific - (-80) °C)
Fridge (Whirlpool - 4°C)
Incubator 37°C + 5% CO2 (Forma Scientific - S/N 31304-941
Light Microscope (Nikon - Model TMS-F)
pH Meter (Mettler Toledo)
Spectrophotometer (Uvmini - 1240 Shimadzu - S/N A109346)
Starstadt tissue culture flasks - T75cm, T25cm.
Sterile Pipettes - 1ml, 5ml, 10ml.
Water bath 37°C (Grant - S/N 619749010)
Waste container
70% Ethanol
Note: All cell culture was undertaken in microbiological safety cabinet using aseptic technique (spraying of 70% ethanol before and after use), to ensure sterility, preventing contamination of the cells. All glassware and other containers used within cabinet were sprayed with ethanol. Gloves were worn at all times. Sterility testing was carried out on media and PBS, every 2days.
