User:Kinkreet/Immunology/Tools used in Immunology

Cell sorting
Stem cells and progenitor cells express different cell-surface markers; in a cell suspension, there is often a mixture of all these cell types, what is often useful is to separate different cell types based on their surface markers (looking at multiple characteristics) so we can 1) quantify the proportion of each cell type, 2) allowing us to study/use one cell type at a time and 3) allow us to measure the differentiation of stem cells with time. These can be done using flow cytometry. Flow cytometry can also be used on particles, chromosome, beads, bacteria and other cell types; clinicians uses flow cytometry to determine the percentage of CD4+ T helper cells in patients with HIV/AIDS, to monitor the status of their immune system.

The cells are first suspended and diluted so that each droplet effectively contain only one cell, the size of the particle usually should be between 0.2-50μm, so anything ranging from a bacteria to a macrophage is suitable. The drops are then passed through a narrow corridor, through an area called 'interrogation point', where the cells will potentially encounter a laser beam. If there is a cell in the drop, it will flow through a laser beam (consisting of different coloured-beams, shaped by prisms and lenses), and absorb some of the light (creating a shadow, called forward scatter or FSC), as well and dispersion of the incident light (called side scatter or SSC) and fluoresce if the particle/cell is fluorescent. The forward scatter is measured jsut off the axis of the incident light, and measures the absorbance by the cell of different wavelengths of light, and thus the detectors (photodiode) will have sensitivity to the wavelengths of the incident light; forward scatter meansures size and refractive indices - the larger the cell, the bigger the FSC; the larger the difference between the refractive indices of the cell to the fluid, the bigger the FSC. Side scatter occurs because of the dispersion, reflection and diffraction of light as the light passes through the cell; side scatter are collected through various lenses located approximately 90° to the axis of the laser beam, where the lenses direct the scatter to an optical fibre which passes into an octagon of detectors, where each wavelength is absorbed one at a time to measure the different intensity of each wavelength in the side scatter. Side scatter measures the internal complexity of the cell, as more granual cells (such as granulocytes, compared to progenitor and stem cells) will exhibit higher side scattering.

Apart from measuring physical characteristics of the cell, one can also measure the abundance of surface markers on these cell types using fluorophore-conjugated antibodies. The antibodies will bind to the surface marker antigen, and when passed under the laser beam, will fluoresce; the more markers there are, the brighter the fluorescence. This fluorescence can be measured and plotted in much of the same way as for FSC and SSC, and the cells isolated for further study. For example, HSC LT-HSC expresses CD150 but not CD48, CD34 nor Flk2. All HSC expresses Kit and Sca, while all lymphocytes do not express Lin.

If the whole population in a cell suspension is analysed, there are often many overlaps in the plots between different cell types on fluorescent plots, and so to improve on the data, a technique called gating is used. Gating set a characteristic boundary on the FCS/SCC plot to decide what cells are analysed. This limits of number of events recording to those which are of interests to us. First, the cell counts which exhibit high FSC and SSC are post likely to be cell doublets, and those with a low FSC but relatively high SSC are thought to be cell debris; both are gated out first, to obtain single cells. After this primary gating, the selected populations can be ran again to separate out different cell types based on granularity and cell size. A second gate can be applied here to select out a single cell type, which can be analysed using fluorescence, to identify subsets within the population, either looking for markers to identify differentiated cells, or for the lack of markers to isolate HSCs. Multiple gates can be used and plotted in different colours so the areas of overlap can be observed, while maintaining all the data points. A third or fourth gate using antibodies against different cell surface markers will often give even more distinct results.

Apart from looking at cell surface markers, we can also use alternative methods, such as using Hoechst dyes. Stem cells have an ABC transporter which would normally be able to pump out these dyes, and so flow cytometry can be used to separate out the cells with a high absorbance due to the dye (differentiated cells) from the low absorbance (HSC cells). The dyes sused are Hoechst 33342, rhodamine and Hoeschst and prpidium iodide.

After the light signal is received by the detector, it is converted to an electronic signal and is then plotted on a graph. If the cell is of interest, it will be moved to a compartment called the 'sheath', otherwise, the cell will simply flow to waste.

HSC transplantation
HSC are hard to obtain, and even though we can identify them using cell surface markers, the only accurate way of knowing is by bone marrow transplantation, where we inject the suspected HSC into the bone of mice, to see if it can create all haematopoietic cell over time. But injecting a full bone marrow would not make a difference, as the HSC marrow in the bone would simply carry on its function; and so the recipient must be conditioned by receiving a high dose of chemotherapy agent and γ radiation, to wipe out the existing HSC. After this conditioning, the sample HSC is added in and wait for 3-4 weeks to see if the HSC cell can regenerate the whole immune system. We monitor this by monitoring the components of the peripheral blood, and also optically, as the bone marrow will go white.

HSC transplantation allows us to kill cancer tumour cells using high does of radiation or chemotherapy agents, and then regenerate a healthy immune system by injecting with HSC, taken out from the same patient before the treatment (using mobilising agents such as G-CSF, or biopsy on hip bones or sternum), back into the blood; the stem cells will find their way to the bone marrow by themselves and re-establish new blood cells. Furthermore, if the bone marrow is not from yourself (in a allogeneic transplantation, as opposed to an autologous transplantation), it may have receptors against the tumour cells, and thus might even be able to actively destroy the tumour cell; a drawback of this is that it may also attack healthy host cells, leading to autoimmune diseases. However, in autologous transplantation, the treatment might not fully cure the disease, potentially allowing the more potent tumour cells to remain and proliferate further; furthermore, some of the tumour cells in the patient may remain and re-establish itself if introduced back.

A hybrid may also be possible, where the donor stem cells are first reacted with a biopsy of the host cells, and any cells which did not react are added in along with the host HSC, allowing for less autoreactivity but better effectiveness against tumour cells.

Suicide genes that encodes for a toxin can also be introduced, and they do not act unless administered with a drug (possibly a transcription factor).

Genomic tools
Inducing mutations in mice is a common way to study the function of a gene; the most common model for study is the mouse. The mouse model have several advantages, for example it is small in size (compared to other models such as rat, rabbit or swine), high rate of reproduction (21 days gestation, 5 week to fully mature sexually), the whole genome of the mouse is known, with 99% of them having counterparts in humans. Naturally occuring mutation models include SCID mouse, which have a deficiency in the DNA rearrangement of the V/D/J segments, probably due to mutations in the Artemis protein or DNA-PK, contain very few mature B and T lymphocytes. Another is the nude mouse, which lacks the Foxn1 gene, the gene for the transcription factor Wnt, which leads to the thymus not developing, and thus the T cells do not mature from the progenitors released from the bone marrow.

Transgenic
A transgenic mouse is one in which artificial genetic material is permanently introduced into the mouse. The material can be the code for a fluorescent protein, used for visualisation, or a drug, which when the promoter is activated, will transcribe that drug. Genetic insertion can be done in a random fashion, where multiple copies may integrate into the genome, often in a head-to-tail fashion; or in a targeted fashion, where the DNA is introduced into the embryonic stem cells, and selecting only those which has undergone homologous recombination.

The gene inserted into the genome can be expressed by adding in or inducing the transcription of the transcription factors for the promoter of that gene. The insert can also be designed so that the promoter is specific to a particular cell type, for example cD11c is most highly expressed in dendritic cells, and thus will affect dendritic cells the most.

TCR-Tg
T cell receptor (TCR) transgenic (Tg) mice have proven to be a good model for studying T cell development and selection. They are made by injecting the oocyte (before the formation of a nucleus) with the transgene of interest; the oocyte is then inplanted into a foster mother which will give birth to the transgenic animal. The first αβ TCR recognized the male-specific HY antigen, a histocompatibility antigen expressed on the membrane of most male cells; the HY antigen is rejected by female counterparts from the same inbred strain, as well as from cytotoxic T cells and antibodies.

Knockout
Knockout (and knock-in) mouse are where a specific site in the genome is targeted. Knockout means a target gene is disrupted, and thus no protein product is formed; knock-in means a target gene has an insertion mutation.

The DNA construct is inserted into the embryonic stem cells with leukemia inhibitory factor (LIF, a.k.a. IL-6), which prevent the ES cells from differentiating. The knockout/in cells are then micro-injected (using a micro-manipulator) into the blastocyst. The mouse will then grow up to be a chimeric, but because the stem cells are of the altered DNA construct, it will grow up further into a knockout/in mouse. Traditionally, to knockout a gene, you add another indicator gene (antibiotic-resistance, for example) into the exon of that gene, so as to disrupt it.