T cells are specialized white blood cells that recognize and eliminate threats within the body. Flow cytometry is a powerful technology for detailed analysis of T cells. This method allows researchers to identify, count, and characterize different T cell populations, providing insights into immune responses.
T Cells and Their Immune Role
T cells, or T lymphocytes, are white blood cells that develop in the bone marrow and mature in the thymus. During maturation, T cells undergo a selection process to ensure they can identify foreign invaders without attacking healthy cells. Once mature, T cells circulate in the bloodstream and lymph tissues, such as the spleen and lymph nodes, to respond to infections and abnormal cells.
T cells are central to adaptive immunity, the body’s targeted defense against specific pathogens. Cytotoxic T cells, identified by the CD8 protein (CD8+ T cells), directly kill cells infected with viruses or bacteria, and destroy cancer cells.
Helper T cells, marked by the CD4 protein (CD4+ T cells), coordinate the immune response by secreting cytokines. These activate other immune cells, including cytotoxic T cells, B cells (which produce antibodies), and macrophages. Regulatory T cells, also known as suppressor cells, prevent the immune system from overreacting and attacking healthy tissues, maintaining immune tolerance.
How Flow Cytometry Works
Flow cytometry measures characteristics of individual cells as they flow in a single stream through a laser beam. Cells are suspended in sheath fluid, which ensures they pass through the instrument one by one in a narrow stream.
As each cell passes through the laser, it scatters light. Two main types of light scatter are measured: forward scatter (FSC), which indicates cell size, and side scatter (SSC), which indicates internal complexity or granularity. Detectors capture these signals, converting them into electrical pulses.
To identify specific cell types or characteristics, cells are often labeled with fluorescently tagged antibodies. These antibodies bind to unique proteins on the cell surface or inside the cell. When the laser excites these fluorescent tags, they emit light at specific wavelengths, which is detected by photomultiplier tubes (PMTs). The intensity of this emitted fluorescence indicates the amount of the target protein present on or within the cell.
Applying Flow Cytometry to T Cells
Flow cytometry is well-suited for studying T cells because it identifies and quantifies different T cell subsets based on their unique surface markers. Antibodies conjugated to various fluorescent dyes target specific cluster of differentiation (CD) proteins expressed on T cell membranes. For example, CD3 is a universal marker for all T cells, while CD4 distinguishes helper T cells, and CD8 identifies cytotoxic T cells.
By using panels of multiple fluorescent antibodies, researchers can differentiate between naive, activated, or memory T cells, and even assess their activation states. For example, markers like CD25, CD69, CD38, CD134 (OX40), and CD137 (4-1BB) are used to identify activated T cells. This detailed characterization is useful in various clinical and research applications.
In clinical diagnostics, flow cytometry is used to:
Monitor immune deficiencies, such as tracking CD4+ T cell counts in individuals with HIV/AIDS, which helps assess disease progression and treatment effectiveness.
Aid in monitoring autoimmune diseases by evaluating changes in specific T cell populations.
Assess transplant rejection by identifying immune cells attacking the transplanted organ.
Serve as a tool in cancer immunotherapy research, helping characterize T cells that infiltrate tumors or are engineered for therapeutic purposes, such as in CAR T-cell therapy.
Interpreting Flow Cytometry Results
The output of a flow cytometry experiment appears as graphs, such as histograms or dot plots. Histograms display single-parameter data, showing the fluorescence intensity of a specific marker on the x-axis and the number of cells (count) on the y-axis. A shift in the peak of a histogram to the right indicates an increase in the expression of the targeted marker.
Dot plots, also known as scatter plots, are used to visualize two parameters simultaneously, with one parameter on the x-axis and another on the y-axis. Each dot represents an individual cell, and distinct clusters of dots indicate different cell populations based on their combined expression of the two markers. For example, a dot plot showing CD3 versus CD4 expression delineates CD4+ T cells from other cell types.
Researchers use “gating” to draw boundaries around these clusters on dot plots, to quantify the percentage or number of cells within a specific population. The mean fluorescence intensity (MFI) of a population provides a measure of the average expression level of a particular marker. Interpreting these results involves comparing the observed percentages or MFIs to established healthy ranges or baseline measurements from the same individual over time. This comparison helps scientists and clinicians understand changes in T cell populations, which can indicate immune system health, disease activity, or response to treatment.