University of Virginia Health System

Small Lymphocyte

a. Small lymphocyte- Peripheral blood smear, Wright-Giemsa stain, 1000x

b. Spindle-shaped lymphocyte- Peripheral blood smear, Wright-Giemsa stain, 1000x

Description:

Although small lymphocytes appear homogenous by light microscopy on a Wright-Giemsa stained peripheral blood smear, they represent cells not only of different lineages but also different stages of differentiation. Their size is somewhat variable (8-10 μm in diameter). Most are round to slightly oval (a), but some may be spindle-shaped (b), and have either a smooth or slightly irregular cell surface with blebs and spicules. The nuclear:cytoplasmic ratio is high. The nucleus is round, and may be slightly indented and is often eccentrically located. The nuclear chromatin is dense and clumped and nucleoli are not visible even though nucleoli are present. The cytoplasm is scant with a color that ranges from grey to different shades of blue. The various subsets of small lymphocytes in the peripheral blood are determined by immunophenotypic marker studies. The range of normal values for the different subsets is great. Most are T cells (50-80%) with a helper (CD4+):suppressor (CD8+) ratio of approximately ~2:1. The majority have the αβ T cell receptor (TCR), but a small percent express the γδ receptor (<10%). The range of normal values for B cells (CD19+) is also great (5-20%), and the κ:λ ratio is approximately 2:1. A subset of normal B cells also express the T cell marker, CD5, and are referred to as B1a cells in the murine system, and are felt to be of fetal liver origin. B1b cells and B2 cells (conventional B cells) do not express CD5, and are of bone marrow origin. Stem cells (CD34+) represent less than 1%. Several other immunophenotypic subsets are often identified (e.g. T cells coexpressing CD4 and CD8, T cells negative for both CD4 and CD8), and together they usually represent only about 1% of the small lymphocytes in the lymphocyte gate.

Development, Function and Trafficking:

T cells- Antigen-independent T cell development occurs in the thymus. In congenitally athymic individuals (DiGeorge Syndrome), T cell development does not occur. Even though the thymus gland involutes to a great extent after puberty, T cell development in the thymus continues well into and possibly throughout adult life. Thymic progenitor cells that enter the thymus from the bone marrow are CD34+, CD7+, and CD10+. These cells differentiate into early thymic progenitor (ETP) cells that express the additional marker Il-7Rα and begin to express the pan T cell markers, CD5 and CD2. The ETP cells are not yet restricted to the T cell lineage, and can still differentiate into dendritic cells and NK cells. At the next stage, the pre-T cell stage, CD1a is expressed along with CD2 and CD5, and they still express CD34 and CD7. pre-T cells are restricted to development down the T cell lineage. pre-T cells next express CD4 and are referred to as CD4+ immature single positive (ISP) T cells. The αβ TCR and CD3 start to be expressed. CD8 expression occurs next leading to a CD4 and CD8 double positive (DP) stage that now expresses the αβ T cell receptor and CD3. From this stage cells develop that are either CD4+ or CD8+ näive T cells that are then released into the circulation and home to T cell areas in peripheral lymphoid tissues. The γδ T cells develop from pre-T cells, and are double negative cells (CD4 and CD8 negative).

B cells- Early antigen-independent B cell development in children and adults occurs in the bone marrow. During prenatal life early B cell development occurs in the fetal liver and the omentum. B cells develop from CD34+ human stem cells through the CD34+, CD10+ common lymphoid progenitor (CLP) cells to early B cells that express CD79a, one of several pan B markers (others include CD19 and Pax5) that are expressed at all stages of development except at the terminal mature plasma cell stage. These early B cells still express CD34 and CD10. The next stage is the pro B cells that express CD19, Pax5, TdT, and RAG while still expressing CD34 and CD10. These pro-B cells differentiate into pre-B cells that are characterized by the presence of cytoplasmic mu (μ) heavy chains that are formed following rearrangement of the immunoglobulin heavy chain genes. At this stage CD34 and TdT are no longer expressed, but CD10 and RAG are still expressed. The pre-B cell receptor (pre-BCR) appears briefly on the cell surface during this stage of development. The structure of this early receptor is that of two mu heavy chains and two surrogate light chains. The surrogate light chains are later replaced by kappa light chains that are formed following rearragement of the immunoglobulin light chain genes to form the mature B cell receptor (BCR). The next two stages are referred to as immature B and mature B cells. The immature B cells express sIgM, and a mature BCR. The mature (näive/,virgin) B cells express both sIgM and sIgD, and no longer express RAG or CD10. The näive or mature B cells are released from the bone marrow and home to B cell areas (primary and secondary follicles B cell follicles) of peripheral lymphoid tissue.

Entry of circulating leukocytes into the various tissues and organs involves several sequential steps referred to as tethering, rolling, activation, adhesion, and diapedesis. Tethering refers to the slowing down of the speed of leukocytes after they make contact with surface endothelial molecules. With näive lymphocytes, the interaction is between selectins on the lymphocyte surface and selectin ligands on the surface of the endothelial cells of the high endothelial venules (HEV) of lymph nodes. As a result of these interactions, the lymphocytes start to roll along the endothelial surface at a much lower rate of speed. Chemoattractants produced locally activate integrins on the lymphocyte surface resulting in tight binding to the integrin ligands on the endothelial cells. Then, through a process called diapedesis, the cells migrate through the space between the individual endothelial cells into the subendothelial matrix of tissues and organs. Once näive lymphocytes enter lymph nodes, T cells home mostly to interfollicular areas and B cells to primary lymphoid follicles or to the mantle zones of secondary lymphoid follicles where antigenic stimulation occurs. In the spleen, näive lymphocytes exit the circulation into the marginal zones, and then into the white pulp. Once in the white pump näive B cells home to B cell follicles and näive T cells to the periarteriolar sheaths. The homing signals probably differ in structure depending on the specific organ (e.g. lymph node vs. Peyer's patches in the intestine).

Both T cells and B cells function in the adaptive immune response. Once released from the thymus and bone marrow, näive or virgin T and B cells, unlike natural killer cells, must undergo antigen stimulation and further proliferation and differentiation to function in the adaptive immune response. Once cloned, T cells function through a cell-mediated immune response while B cells either terminally differentiate into plasma cells which produce antibodies (immunoglobulins, gammaglobulins) which are the immune mediators (humoral immune response), or become memory B cells that recognize a second challenge from the same antigen resulting in the anamnestic response.  

Näive T cells require antigen presentation predominantly from dendritic cells that have engulfed and processed pathogens from various tissues and organs. After engulfing and processing pathogens, immature dendritic cells (iDCs) then exit these sites through afferent lymphatics. During this migration, they mature and express human leukocyte antigens (HLAs) on their surface that are important in the presentation of antigen to näive T cells. The antigen-specific CD4+ helper T cells of the Thy 2 subset that are crucial to the T cell-dependent B cell development in the germinal center are generated by these same dendritic cells through their MHC class II molecules. This interaction between cells of the innate immune system, in this case dendritic cells, is referred to as the "immunological synapse". Näive B cells also may be stimulated directly through antigen presentation by dendritic cells, but most B cells probably are stimulated by soluble antigen. Regardless, the antigen-specific CD4+ Thy 2 cells are needed for the further differentiation of B cells to plasma cells or memory B cells in the germinal center. Memory or committed B and T cells that emerge as a result of antigen stimulation leave the secondary lymphoid organs through efferent lymphatics into larger lymphatic vessels such as the thoracic duct which empties into the left subclavian vein. Circulating memory lymphocytes then home to the specific tissues or organs where the initial contact between pathogens and dendritic cells occured. This remarkable homing property is conferred upon memory lymphocytes by dendritic cells specific for a particular tissue and organ. Memory lymphocytes make up probably 40-50% of the circulating lymphocytes. The range of lifespan of lymphocytes is quite variable, depending on lineage and level of differentiation and activation. Most live 3-5 years, but some live much longer.

General References:

1. Larosa CV, Stadtfeld M, Graf T. Determinant of lymphoid-myeloid lineage diversification. Annu Rev Immunal 2006; 24:705-38
2. Blom B, Spits H. Development of human lymphoid cells. Annu Rev Immunal 2006; 24:287-320
3. McHeyzer-Williams LJ, McHeyzer MG. Antigen-specific memory B cell development. Annu Rev Immunal 2005; 23:487-513

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