Immunophenotyping of lymphocyte subpopulations
Cell identification is based on detecting specific marker antigens using fluorochrome-labeled antibodies.
- T lymphocytes: the total population is counted using monoclonal antibodies against CD3, the absolute T-cell marker. The CD4+ and CD8+ subpopulations are identified alongside CD3 using fluorochromes with contrasting emission (green, orange, pink), since these receptors can also be expressed on other cells.
- B lymphocytes: identified using antibodies against CD19 (present on all B cells). To assess clonality in lymphoproliferative disorders, membrane $\kappa$ and $\lambda$ chains are additionally investigated.
Assessment of proliferative activity and cell cycle
The functional capacity of T cells is assessed by their polyclonal proliferative response to mitogens. In modern practice, stimulation is performed using plastic-bound monoclonal antibodies to CD3 combined with soluble antibodies to the costimulatory molecule CD28.
The following approaches are used to measure proliferation and cell cycle parameters:
- Flow cytometry with CFSE labeling: the carboxyfluorescein succinimidyl ester dye binds to intracellular proteins and is evenly distributed between daughter structures during mitosis. On the cytogram, the rightmost peak is formed by undivided cells, while peaks shifted from right to left reflect populations that have undergone 1, 2, 3, or more divisions.
- Radioisotope method: measuring $^3$H-thymidine incorporation serves as the sensitivity gold standard, though it is associated with the inconvenience of using radionuclides.
- Cell cycle assessment: propidium iodide staining using specialized software allows calculation of the fraction of cells with hyperdiploid DNA content in the G1+S, G2, and mitotic phases.
Laboratory diagnostics of apoptosis
Analysis of programmed lymphocyte death is performed using two cytofluorimetric methods:
- Propidium iodide staining: during apoptosis, part of the nuclear material is lost, so dying cells form a characteristic hypodiploid peak located to the left of the diploid peak on the histogram.
- Annexin V binding: labeled annexin V selectively binds to phosphatidylserine, which flips to the outer surface of the cell membrane exclusively during apoptosis.
Functional analysis of T helpers (Th1 and Th2) and ELISPOT
Assessing the capacity of T helpers to produce cytokines requires intracellular staining and includes consecutive steps:
- Activation: cells are stimulated with a combination of phorbol myristate acetate (a protein kinase C activator) and ionomycin (a calcium ionophore) bypassing antigen receptors.
- Secretion blockade: the dye brefeldin A disrupts intracellular transport in the Golgi apparatus, trapping cytokines inside.
- Permeabilization and staining: the membrane is treated with saponin, after which antibodies to cytokines are introduced. Key markers are IFNγ for Th1, and IL-4 for Th2.
To quantify cytokine-secreting cells (predominantly IFNγ producers of Th1), the ELISPOT (Enzyme-linked immunospot) method is also used. In this assay, cells are incubated on a nitrocellulose membrane coated with antibodies, capturing the secreted cytokine via enzyme immunoassay.
Briefly: answers to topical questions
On which immune cells, besides T lymphocytes, can CD4 and CD8 receptors be expressed?
In addition to T lymphocytes, CD4 and CD8 receptors can be expressed on macrophages and dendritic cells.
- CD4 is present in small amounts on some macrophages, making them susceptible to human immunodeficiency virus infection.
- CD8 is used to analyze subpopulations in mice and marks a significant portion of lymphoid dendritic cells.
What is the principle of visualization and quantification of secreting cells in the ELISPOT assay?
The principle of visualization and quantification in ELISPOT is based on the enzyme-linked detection of locally secreted cytokines.
- Visualization — the cytokine secreted by the cell binds to antibodies fixed on the nitrocellulose membrane. The resulting complex is detected using secondary antibodies labeled with peroxidase, leading to the formation of colored spots.
- Quantification — performed by counting the formed colored spots using a microscope or a specialized device. Each spot corresponds to a single cell secreting the target cytokine.
Which surface differentiation antigens are used as markers of early and late T-lymphocyte activation?
The following surface antigens are used as markers of T-lymphocyte activation:
- CD69 — the earliest activation antigen, appearing on the surface within 2–3 hours.
- CD25 — an early activation marker representing the $\alpha$ chain of the interleukin-2 receptor (appears after 4 hours).
- CD71 — an intermediate-stage marker, the transferrin receptor (expressed after 24 hours).
- MHC-II — late markers of T-cell activation, whose expression begins on days 3–6.
Adhesion molecules ($\beta_1$ integrins) also appear at a late stage (days 4–8).
CD3 — ALL T cells; CD19 — ALL B cells; CFSE — counts divisions from right to left; Annexin V — detects phosphatidylserine in apoptosis.
Class and exam questions
Why are CD4 and CD8 identified only in combination with CD3?
CD4 and CD8 molecules can be expressed not only on T lymphocytes. To accurately identify T-cell subpopulations, they are stained simultaneously with CD3 using contrasting fluorochromes.
How to interpret the cytometry graph when using the CFSE dye?
The rightmost peak with the highest fluorescence intensity represents undivided cells. Each subsequent peak to the left corresponds to cells that have undergone 1, 2, 3, or more division cycles.
Why are brefeldin A and saponin used in cytokine analysis?
Brefeldin A blocks the transport of substances through the Golgi apparatus and prevents the outward secretion of cytokines. Saponin provides membrane permeabilization, allowing antibodies to penetrate inside the cell.
What else to study on this topic
- Using membrane kappa and lambda chains to assess B-cell clonality
- Comparative analysis of the radioisotope method ($^3$H-thymidine) and CFSE labeling
- Assessment of cell clone reactivity in the leukocyte migration inhibition assay
- Detailed protocol for performing and detecting results in the ELISPOT assay
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