There are three major types of lymphoid cells: B-cells, T-cells, and Natural Killer (NK) cells. B-cells develop in the bone marrow and are responsible for production of immunoglobulins (a.k.a. antibodies). T-cells develop in the thymus. NK cells arise from bone marrow and are stimulated by interferon to recognize and kill "non-self" cells including microorganisms and cancer cells.
All lymphocytes descend from a progenitor lymphoid stem cell, which gives rise to a lymphoid blast, which can be recognized on light microscopy by virtue of being larger than a mature lymphocyte and bearing a high nuclear:cytoplasm ratio and prominent nucleoli in the nucleus. B cell development in the bone marrow is highly coordinated, beginning with creation of a pro-B cell and culminating in a mature B cell. A number of different cell surface markers known as Cluster of Differentiation (CD) proteins are produced during B cell development; these CD proteins characterize the different stages of B cell maturation. Also during this time, the immunoglobulin (Ig) genes that will eventually encode the mature antibodies undergo a series of complex gene rearrangements via a process known as V(D)J recombination. The end result is a mature B cell that contains rearranged Ig genes encoding a transmembrane Ig protein on the cell surface that acts as the B cell receptor for foreign antigens. Once the mature B cell becomes activated by binding of the B cell receptor to a specific antigen (as occurs in the setting of an active infection), it differentiates into two types of cells: a plasma cell, which is a terminally-differentiated effector B cell whose major function is to secrete antibodies, and a memory B cell, which serves as a long-term genetic reservoir of immunologic memory in the event of a future infection by the same microbe.
Some of the major CD proteins in B cell development are CD19 and CD20, which are produced throughout much of B cell development; CD10, which is a marker of the part of a lymph node known as the germinal center (described further below); and CD38, which is expressed in plasma cells.
Like B cell development, T cell development also proceeds via a series of highly complex, regulated steps, beginning with a pro-T cell and ending in a mature T cell. The T cells express a variety of different CD markers at different stages of maturation, most notably CD4 or CD8, which identify two different types of T cells. CD4+ T cells are known as helper T cells as they assist in B cell receptor-antigen interactions. CD8+ T cells are known as cytotoxic T cells as they function to kill other cell types by triggering apoptosis.
Some of the major CD proteins in T cell development are CD3, which is present on all T cells; CD4, which identifies CD4+ helper T cells; and CD8, which identifies CD8+ cytotoxic T cells. Clinically, CD4 and CD8 are important in patients with HIV, which infects CD4+ T cells, leading to a reduction in CD4+ to CD8+ T cell ratio (which under normal circumstances is around 2:1).
B and T lymphocytes appear identical under light microscopy. Each is ~7-15 µn;m, with blue cytoplasm and an ovoid nucleus containing coarse chromatin. A representative lymphocyte is shown below.
Whereas B and T cells are part of the adaptive immune system (i.e., the immune system that governs antigen-specific immune responses), NK cells are part of the innate immune system in that they serve to provide general, rather than antigen-specific, immunity. They express CD16 and CD56 on their cell surfaces and are negative for the T cell marker CD3. Morphologically they are large, unusual-appearing lymphocytes, a representative of which is shown below.
T-cells, B-cells, and NK-cells can also be differentiated from one another by using flow cytometry to identify their proteins and cell surface markers. In this technique, cells are stained with fluoroscopic markers that each have specificity against a different cellular protein. The stained sample is then run through a machine that exposes the cells to a laser; any cells bearing the specific protein(s) in question will fluoresce, and the different wavelengths at which the cells fluoresce allow for identification of multiple different cell types. Consider, for example, the following flow cytometry plot
This shows one population of cells that express CD19, a pan-B cell marker, plus CD5, a T cell marker, in addition to a smaller population of cells that are CD5+ CD19-.
A basic understanding of the lymphoid system is essential in order to understand the various lymphocyte disorders. The lymphoid system contains primary and secondary lymphoid organs. Primary lymphoid organs include the bone marrow, where B cell development occurs, and the thymus, the site of T cell development. Secondary lymphoid organs include the lymph nodes, mucosa-associated lymphoid tissue (MALT), and the spleen. The secondary lymphoid organs are the sites where antigenic stimulation of B cells occurs. There are hundreds of lymph nodes disseminated all over the body. Each has a defined structure, consisting of primary lymphoid follicles where mature non-activated B cell congregate, and secondary lymphoid follicles (a.k.a. germinal centers), which arise when B cells in primary follicles become activated by antigenic stimulation. A schematic of a typical lymph node is shown here.
The orderly architecture of the lymph node often (but not always) becomes disrupted if the lymph node is infiltrated by a malignancy of the lymphoid cells. Such malignancies may be categorized as leukemia or lymphoma. Historically, leukemia was invoked to describe a condition in which the malignant cells were presently primarily in the blood and bone marrow, while lymphoma was used to describe a lymphoid malignancy where the malignant cells primarily homed to the lymph nodes, spleen, and peripheral lymphoid tissues. We now know that this is an oversimplification, since many patients with lymphoma will have malignant cells in the blood and/or bone marrow, and many with leukemia will have lymph node and splenic involvement Ð a classic example being chronic lymphocytic leukemia (CLL), an indolent B cell disorder that can sometimes be present exclusively in the blood and bone marrow (where it is referred to as CLL) and other times be present largely in the lymph nodes (where it is referred to as small lymphocytic lymphoma (SLL)). The World Health Organization (WHO) circumvents this problem by categorizing the lymphoid malignancies according to the point at which lymphoid cell development becomes aberrant during lymphoid development, leading to malignancy. According to this designation, most acute lymphoid leukemias such as acute lymphoblastic leukemia will have a defect in early lymphoid differentiation, while most lymphomas will have a defect in mature lymphoid cells. CLL/SLL are categorized together as a mature B cell malignancy in the WHO classification.
A common diagnostic problem involving lymphocytes that many physicians face at some point in their careers, regardless of specialty, is the evaluation of a patient with lymphocytosis, defined as an increase in absolute lymphocyte count, i.e., the percentage of lymphocytes multiplied by the total white blood cell (WBC) count. As we learned for myeloid cells, red cells, and platelets, the approach to lymphocytosis begins with categorization as either primary (due to a bone marrow or other primary hematologic disease) or secondary (due to a stimulus exogenous to the marrow). These categories can be distinguished on the basis of the peripheral blood smear, history, and sometimes flow cytometry. As an example, consider a college student who presents to urgent care clinic with two weeks of fever, profound malaise, a sore throat, and swollen cervical lymph nodes, who has a complete blood count (CBC) drawn that shows an elevated lymphocyte count, and whose blood chemistries reveal an increase in liver function tests. What would the diagnosis be if the following were seen on the smear?
The answer is: infectious mononucleosis. The cell above is called an atypical lymphocyte, so-named because it has certain features of a lymphocyte (i.e., blue cytoplasm, absence of significant nuclear folding) but clearly looks atypical from a normal lymphocyte based on the large size of the cell and the "scalloping" of the plasma membrane against adjacent red blood cells. This is a hallmark of viral infections. In the context of a young patient with concomitant cervical lymphadenopathy, malaise, and transaminase elevation, EBV infection is the most likely cause.
Alternately, consider the case of an elderly man who is found to have an elevation in lymphocyte count, diffuse lymphadenopathy, and the following smear. What is the likely diagnosis?
The answer is: chronic lymphocytic leukemia (CLL). This is a B cell lymphoproliferative disorder that is due to malignant B cells, which have a peculiar immunophenotype in that they express CD19 (a pan-B cell marker) and also CD5 (a T cell marker), and they are dim for CD20 (whereas most B cells have robust CD20 expression). The smear contains a preponderance of mature-appearing malignant lymphoid cells, along with large, irregular-appearing cells on the smear; these latter cells are called smudge cells and represent malignant lymphocytes that have been damaged by the smear preparation process. The constellation of asymptomatic lymphocytosis in an elderly person with diffuse lymphadenopathy and the above smear is very characteristic of CLL, although flow cytometry would clinch the diagnosis.