T cell Function
The major function the T cell, particularly the T helper cells having CD4+ receptors regulate the humoral immunity by playing a major role in the stimulation of B-cells. The overall development, maturation, differentiation as well as antibody secretion is stimulated by T helper cells. The whole process is governed controlling the transcription factors responsible for B-cell development that are determined by the position of the germinal centres (Crotty 2014). The process of antigen presentation that occurs when a foreign particle invades a living body that starts a chain reaction of immunological responses, autoimmune disorders as well as cancer is controlled by T cells. The differentiation of B-cells is controlled by T cells by activating chemokine receptors that directs the effector cells towards the inflammation or infectious site.
The function of the other type of T cells that is the Cytotoxic T cells are generally directly related with antigen processing and destruction (Blum Wearsch and Cresswell 2013). In exposure to such an infection the cytotoxic T cells release various cytotoxic substances like perforin, granulysin, granzymes et cetra. These chemical substances invade the antigen cell and activate the serine protease function to activate the caspase activated apoptotic cascade (Bakshi Cox and Zajac 2014). The cytotoxic T cells are also stimulated by T helper cells. Cytotoxic T cells also release cytokines such as TNF-α and IFN-γ, which provide immunity against intracellular pathogens as well as have function in cancer inhibition.
Development of tumour occurs by evading the immune response of the affected host. The condition forms a suppressed immune response condition by weakening the cytological responses and down regulating the effecter cells near the tumour. T cells regulate a major part of the cytological responses and generating B-cells as discussed above. This is why scientists are developing ways to modify the effects of the T cells of the affected cancer patient as a therapeutic strategy. The modification is targeted to alter the receptors on the T cell surface or by introducing genes that can recognise antibody in Chimeric Antigen Receptors (CARS) show promising futures (Sharpe and Mount 2015). Particularly, the genetically modified T cells can be used to successfully B cell malignant condition in hematological trials. Many healthcare and medical organizations are forming partnerships to invest more in this particular field of research as of late (Kershaw Westwood Slaney and Darcy 2014). There are although a few shortcomings of this particular therapy, like incorporating the genetically modified T cells into the patient and this kind of problems affect the safety and efficacy standards in clinical trials. The Food and Drug administration or FDA in United States approved to therapies as of 2017. Treatment of Acute Lymphoblastic Leukemia or ALL, in case of treatment amongst children and another for adults with progressive lymphoma condition (National Cancer Institute 2014). The promising clinical data maps the progress in understanding the mechanism of tumour physiology and the improvements that can be used to produce cell products are all in progress with the clinical version of these important cellular immunological therapies, but still scientists are unsure whether this approach will be efficient against breast and colorectal malignancies.
T cell and Cancer Therapy
HIV or Human Immunodeficiency Virus is a kind of retrovirus that causes an autoimmune disorder and infects immune cells in the human body like the T helper cells with CD4+ receptors as well as macrophages and dendrite cells (Altfeld and Gale 2015). HIV infection leads to down regulation of CD4+ T helper cells through various mechanisms, including pyroptosis, which is a process where immune cells detect antigens inside themselves and selectively secrete cytokines that self-destroy, apoptosis of uninfected adjacent cells, directly killing affected cells by lysis (Pham et al. 2014), and killing of affected CD4+ T helper cells by CD8+ cytotoxic T cells that recognize infected cells (Doitsh et al. 2014). Complete loss of immunity occurs when the number of CD4+ T helper cells declines below a critical threshold level, leading to loss of cell-mediated immunity and the body becomes more vulnerable to various opportunistic infections, ultimately leading to the development of AIDS.
Various Co-stimulatory and co-inhibitory receptors control the development and propagation of T cell biology. These receptors determine the positional and functional outcomes of the receptors in T cell surface. The antigen detection, presentation to their respective Major Histocompatibility Complex proteins (Birnbaum et al. 2014), engaging cytotoxic killer cells and activating B-cells is controlled by these receptors that send out cellular signals to relay proteins that carry out specific duties. It is also found in research journals that these receptors are stimulated by one or more receptors that form a crosstalk network to ensure maximum efficiency (Chen and Flies 2013). Many researches have been conducted over the years to find the relation between the co stimulatory effects of the T cells and its effect in disease control.
Prolonged stimulation of T cells during chronic infections and autoimmune diseases can lead to complete exhaustion of these cells. This can lead to persistence of the current infection and the affected person will suffer. Various clinical techniques have been used to completely understand the mechanism of action. Scientists used the expression of favourable surrogate markers that are known to play a role in co-stimulation/exhaustion function in separate data sets, where it was found that these markers could facilitate the receptor action and control the disease condition even during exhaustion as a substitute (McKinney et al. 2015). Positive clinical evidence was found using this response therapy in infective like hepatitis C and vaccination cases like yellow fever, malaria, influenza, but the results autoimmune and inflammatory diseases like diabetes, systemic lupus, idiopathic pulmonary fibrosis and dengue fever was not very successful. It can be concluded that the exhaustion of T cells is very important in the outcome determination of many autoimmune diseases and leads to severity of the disease if not treated properly.
T cell and HIV
The importance of the T lymphocyte discussed clearly signifies its importance in regulating the immunological function an its role in HIV infection, so observation of Lifespan of T cell is very important to implement various cytological therapies to control the function of T cells. The aim of this paper is to address that issue and develop a method to develop in vitro culture of lifespan of T cells.
The aim of this research proposal is to successfully observe the lifespan of T cell in vitro conditions and map the propagation using sophisticated techniques.
Method:
Sample Collection:
Fresh blood samples can be collected from the Blood Bank to ensure the collected sample is disease free and from a healthy individual.
T cell isolation can be done by density gradient centrifugation to derive the Peripheral blood mononuclear cell or PBMC (Higdon Lee Tang and Maltzman 2016). Approximately 400 mL of whole blood sample is transferred to a few 50mL centrifugation tubes. The blood sample is then diluted with Phosphate buffer solution (PBS) in a 1:11 ratio. The mixture is then layered over a Ficoll solution by gentle pipetting method holding the tubes carefully at an angle. This mixture is then subjected to centrifugation until the layers are distinctly separated and PBMC layer is removed dissolved in PBS solution for further assessment.
The PBMC suspension is transferred a T-75 culture flask in 20 mL RPMI 1640 media containing 10% FBS, 1% penicillin/streptomycin, and 1 μg/mL phytohemagglutinin (PHA). This system is incubated at 37°C under 5% CO2 for approx 24 hours for the monocytes to be separated from lymphocytes. The media is removed and subjected to centrifugation to get the T cells in the pellet which is again cultured in a new T-75 flask with RPMI 1640 media and maintain this culture by changing the media every 1-2days.
There are different ways the viability of cells can be detected. One of the most popular methods used in laboratories is the use of tetrazolium compounds, mainly MTT which changes colour of its solution in presence of metabolites (Riss et al. 2016). The T cells are cultured for 24 hours in RPMI 1640 media before MTT assay is carried out. The MTT solution is added to culture mediums containing cells at 0.2 – 0.5mg/ml dilution. This mixture is then incubated for about 4-8 hours in a 96 well format that is subjected to high throughput sceening techniques. The living cells can convert MTT into a purple coloured compound called formazan which is then measured in Spectrophotometer at 570nm to observe changes in absorbance. The reaction mechanism of change of change of MTT to formazan is not properly known, but non-viable cells fail to reduce MTT and no colour change is noticed.
T cell Co-stimulation
FACS or Fluorescence activated cell sorter is a kind of sophisticated flow cytometric machine that detects specific antibody targeted cells which are fluorescently labelled (Shields Reyes and López 2015). To separate T cells from the cell mixture of PBMC, FACS can be used. The sample of T cell culture mixture was treated with NaCl solution and centrifuged and the is then treated with CD4-FITC and CD8-FITC antibody conjugate to separate out the T cells and incubated for some time. A cell suspension is formed and is loaded into a thin stream in the FACS such that the flow rate of the cells become on cell at a time. The cells flow down the stream of liquid and is scanned by a laser.
Study of cell proliferation can be done in many ways including assessment of DNA content during cell division. The widely accepted method of DNA labeling is using a fluorescent dye that will bind to the DNA and will show the amount of DNA present (Tsubouchi et al. 2013). Propidium iodide or PI is such a dye that will bind to the hydrophobic DNA and exude red fluorescence that can be observed in Ultra Violet illuminator at 480 nm. RNase treatment has to be performed before PI treatment on T cells to ensure no interference occurs, because PI binds to both double stranded RNA and DNA. The T cells are fixed with 70% ethanol or detergent solution to make sure all the cells are enucleated. The graphical data will show the change in content of DNA during cell division and that will prove that the cells are healthy and in living condition.
Sample peripheral blood collection from blood bank to ensure the blood is disease free
Lymphocyte Isolation from PBMC by centrifugation to separate all the cellular layers of the blood
T cell culture in RPMI 1640 media which is specific for monocytes and lymphocytes
Checking cell viability using MTT Assay to check the metabolic activity and functionality
T cell separation using FACS using specific antibody to specifically separate T cells
Propidium Iodide Labelling to map cell propagation, which will help map the DNA content that ensure propagation during cell division.
The selected sample is whole blood from blood bank by collecting peripheral blood. This is done so that the sample is disease free and no human or animals are subjected to harming. The preserved blood is from registered voluntary donors.
T cell Exhaustion
The sample size selected will vary with respect to the step and method being used.
Ethical issue can be a hurdle in scientific research and needs to be abided according to the law of the governing body while conducting any scientific experiment. In this case, no animals, or humans would be harmed while performing the research. The blood samples were collected from a blood bank to avoid any direct human collection.
The risk assessment is quite low in this research as the samples collected are disease free. It is suggested that sanitary clinical conditions should be maintained while performing the experiments as it involves cell culture and live cell maintenance. Gloves should be worn under every circumstance to avoid contamination and all the procedures should be performed under supervision.
The research feasibility is according to the rules and regulations of the university and any sophisticated equipment used will be under supervision of the Professor.
References:
Altfeld, M. and Gale Jr, M., 2015. Innate immunity against HIV-1 infection. Nature immunology, 16(6), p.554.
Bakshi, R.K., Cox, M.A. and Zajac, A.J., 2014. Cytotoxic T Lymphocytes. Encyclopedia of Medical Immunology: Autoimmune Diseases, pp.332-342.
Birnbaum, M.E., Mendoza, J.L., Sethi, D.K., Dong, S., Glanville, J., Dobbins, J., Özkan, E., Davis, M.M., Wucherpfennig, K.W. and Garcia, K.C., 2014. Deconstructing the peptide-MHC specificity of T cell recognition. Cell, 157(5), pp.1073-1087.
Blum, J.S., Wearsch, P.A. and Cresswell, P., 2013. Pathways of antigen processing. Annual review of immunology, 31, pp.443-473.
Chen, L. and Flies, D.B., 2013. Molecular mechanisms of T cell co-stimulation and co-inhibition. Nature Reviews Immunology, 13(4), p.227.
Crotty, S., 2014. T follicular helper cell differentiation, function, and roles in disease. Immunity, 41(4), pp.529-542.
Doitsh, G., Galloway, N.L., Geng, X., Yang, Z., Monroe, K.M., Zepeda, O., Hunt, P.W., Hatano, H., Sowinski, S., Muñoz-Arias, I. and Greene, W.C., 2014. Cell death by pyroptosis drives CD4 T-cell depletion in HIV-1 infection. Nature, 505(7484), p.509.
Higdon, L.E., Lee, K., Tang, Q. and Maltzman, J.S., 2016. Virtual global transplant laboratory standard operating procedures for blood collection, PBMC isolation, and storage. Transplantation direct, 2(9).
Kershaw, M.H., Westwood, J.A., Slaney, C.Y. and Darcy, P.K., 2014. Clinical application of genetically modified T cells in cancer therapy. Clinical & translational immunology, 3(5).
McKinney, E.F., Lee, J.C., Jayne, D.R., Lyons, P.A. and Smith, K.G., 2015. T-cell exhaustion, co-stimulation and clinical outcome in autoimmunity and infection. Nature, 523(7562), p.612.
National Cancer Institute. (2014). CAR T Cells: Engineering Immune Cells to Treat Cancer. [online] Available at: https://www.cancer.gov/about-cancer/treatment/research/car-t-cells [Accessed 28 Feb. 2018].
Pham, T.N., Lukhele, S., Hajjar, F., Routy, J.P. and Cohen, É.A., 2014. HIV Nef and Vpu protect HIV-infected CD4+ T cells from antibody-mediated cell lysis through down-modulation of CD4 and BST2. Retrovirology, 11(1), p.15.
Riss, T.L., Moravec, R.A., Niles, A.L., Duellman, S., Benink, H.A., Worzella, T.J. and Minor, L., 2016. Cell viability assays.
Sharpe, M. and Mount, N., 2015. Genetically modified T cells in cancer therapy: opportunities and challenges. Disease models & mechanisms, 8(4), pp.337-350.
Shields IV, C.W., Reyes, C.D. and López, G.P., 2015. Microfluidic cell sorting: a review of the advances in the separation of cells from debulking to rare cell isolation. Lab on a Chip, 15(5), pp.1230-1249.
Tsubouchi, T., Soza-Ried, J., Brown, K., Piccolo, F.M., Cantone, I., Landeira, D., Bagci, H., Hochegger, H., Merkenschlager, M. and Fisher, A.G., 2013. DNA synthesis is required for reprogramming mediated by stem cell fusion. Cell, 152(4), pp.873-883.
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