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Mature lymphocytes can be divided into several functional types and subtypes. The major classes of lymphocytes are the T cells, B cells, and natural killer (NK) cells. T lymphocytes are derived from the thymus (see Chaps. 5 and 76) and are responsible for cell-mediated cytotoxic reactions and for delayed hypersensitivity responses (see Chap. 78). They also produce the cytokines that regulate immune responses and provide helper activity for B cells. B lymphocytes can concentrate and present antigens to T cells and are the precursors of immunoglobulin-secreting plasma cells (see Chap. 77). NK cells account for innate immunity against infectious agents and transformed cells that have altered expression of transplantation antigens (see Chap. 79). This chapter describes methods for isolating lymphocytes and discusses their physical and biochemical properties.

Acronyms and Abbreviations

Acronyms and abbreviations that appear in this chapter include: ADAM, a disintegrin and a metalloprotease; ATP, adenosine 5′-triphosphate; Btk, Bruton tyrosine kinase; DHEA, dehydroepiandrosterone; DNA-PK, DNA-dependent protein kinase; DSBR, double-strand break repair; lck, leukocyte tyrosine kinase; NK, natural killer; RAG, recombination-activating gene; S1P, sphingosine 1-phosphate; TdT, terminal deoxynucleotidyl transferase; ZAP-70, zeta-associated protein of 70 kDa.

Lymphocyte Density

Lymphocytes can be isolated from the blood using density gradient centrifugation. Most commonly, this is performed using a step gradient composed of a mixture of the carbohydrate polymer Ficoll and the dense iodine-containing compound sodium metrizoate.1 This technique takes advantage of the low density of lymphocytes (1.07 g/mL) relative to that of erythrocytes (1.09–1.10 g/mL), granulocytes (1.08–1.09 g/mL), or monocytes (1.08 g/mL).

A Ficoll solution adjusted to a density of 1.077 g/mL is ideal for isolating human lymphocytes. Whole blood is layered onto a cushion of Ficoll-sodium metrizoate prior to centrifugation at 400g for 30 minutes. The denser red blood cells and granulocytes will sediment to the bottom of the tube, and the monocytes will enter into the Ficoll cushion. The lymphocytes can be collected from the interface formed between the Ficoll–sodium metrizoate cushion and the plasma above, which contains the lighter-density platelets (1.04–1.06 g/mL). This layer contains lymphocytes and some monocytes, which can be removed by plating the cells in culture flasks and harvesting the lymphocytes that are not adherent to plastic.

Lymphocyte Surface Antigens

Lymphocyte subsets generally cannot be distinguished from one another by morphology. Most resting lymphocytes appear as small round cells with a dense nucleus and little cytoplasm (see Chap. 74). However, this homogeneous appearance is deceptive, as these cells comprise many functionally distinct subpopulations.

These subsets can be distinguished through the differential expression of cell-surface proteins, each of which can be recognized by a specific monoclonal antibody (see Chap. 15). Coupled with the biochemical analyses of the surface molecules that are recognized by these each of these antibodies, many lymphocyte surface antigens have been defined.

Typically, it is ...

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