The Theory

The discovery of B cells led to a modern theory of antibody production called Clonal Expansion Theory (sometimes still referred to as Clonal Selection Theory). In Clonal Expansion Theory, B and T cells are created with random antibodies, then screened for self-reactivity. When antigen enters the system, it eventually binds to any B cell displaying an antibody specific to that antigen. This binding event triggers the following three steps:

1. Each activated B cell reproduces, to create an expanding population of identical B cell clones. This population is called a clone.
2. Some members of the clone become plasma cells. Plasma cells produce and secrete copies of the antibody displayed on the B cell surface.
3. Other members of the clone are stored as memory cells. Thus, there is a large population of cells to create a strong response when the antigen enters the system again.

Clonal Expansion Theory explains:

1. Specificity, since only antigen-reactive clones are triggered and only antigen-specific antibody is produced. Diversity is not explained but incorporated into this theory.
2. Memory, since clonal expansion explains why subsequent responses to an antigen are exponentially stronger than the initial response.
3. Tolerance, since B and T cells with potential self-reactivity are destroyed or rendered anergic (unable to respond).

Generation of Diversity

An enormous number of specific antibodies and T cells must be generated to respond to the enormous and changing antigenic universe. Immunoglobulin (for B cells) and TCR (for T cells) genes have multiple and similar methods for creating diverse antigenic receptors:

1. Multiple genes encode different protein sequences with different specificities.
2. Multiple segments of genes differentially combine to create increased specificities.
3. Differential junctions following gene segment recombination yield different amino acid sequences.
4. Somatic (after clonal expansion has begun) mutation of receptor genes in various clones causes changes in germline sequences.

Neutrophils are commonly referred to as polymorphonuclear neutrophilic leukocytes (PMNs/PMLs) or polys, even though technically any granulocyte is a PMN. Neutrophils are intensely phagocytic and arrive quickly at infection sites (unhealthy tissues) to phagocytize pathogens. Neutrophils have an F(c) receptor (FCR) which detects antibodies bound to antigens on the surface of pathogens. The process of detecting antibody-antigen complexes is called opsonization. After opsonization, the neutrophil absorbs the infectious microbe and degrades it.

Neutrophils have a lobulated nucleus, meaning it is condensed and dead — hence the term polymorphonuclear. Their cytoplasm is filled with granules containing degradative enzymes such as lysozyme, collagenase, elastase and others. During phagocytosis, these granules combine with phagosomes to break down pathogens.

Neutrophils are abundant in blood, constituting 50-60% of circulating white blood cells. However, neutrophils are absent from healthy (uninfected) tissues and only live about six hours. After performing phagocytosis several times, the neutrophil dies and degranulates (thus releasing its degradative enzymes). These enzymes damage and inflame local tissues — making neutrophils important in inflammation — but these corrosive enzymes also initiate healing.

After understanding Clonal Expansion Theory, it is important to become familiar with the origin and nature of cells involved in the immune systems. Granulocytes and monocytes travel only in the blood. Lymphocytes circulate through both blood and lymph; lymphocytes can exit a lymph node via its efferent vessel, travel through the lymph and then enter the bloodstream via the neck. Cells go back and forth between blood vessels, lymph vessels and tissue via extravasation. Leukocytes (aka white blood cells) are just any cell in the blood other than red blood cells, meaning all the cells below can be white blood cells depending on their location. All blood cells arise via hematopoiesis.

Granulocytes

Granulocytes are a type of white blood cell containing granules. Granulocytes are aka polymorphonuclear neutrophilic leukocytes (PMNs/PMLs) or polys, although PMN can refer specifically to neutrophils since neutrophils are the most common granulocyte. The granules inside granulocytes are secretory vesicle containing a molecule (for example, basophil granules contain histamine). The granules are dormant in the cytoplasm until a cell signal instructs the granules to release their components. This signal is typically shock or distress. Degranulation is when granules release their contents from the cell.

Lymphocytes

Lymphocytes bind to specific antigens and generate from stem cells in primary lymphoid tissue via hematopoiesis. Mφs, dendritic cells and B cells originate in bone marrow; and T cells originate in the thymus. After maturing, lymphocytes migrate to secondary lymphoid tissues (lymph nodes, blood and other tissues) to fulfill their role.

Macrophages

Macrophages (aka mononuclear phagocytes or Mφs) have two main functions: phagocytosis and antigen presentation. In phagocytosis, the macrophage or monocyte cell-surface F(c) Receptor (FcR) binds to the antibody-antigen complex (an antibody bound to an antigen on the cell-surface of a pathogen). The pathogen, antigen and antibody are engulfed and degradative granules and enzymes break it down in the lysosome. Digested antigen fragments are bound to MHC molecules and presented on the macrophage or monocyte cell-surface so that effector cells (B and T cells) can recognize the antigen. Because macrophages present antigens, they are antigen presenting cells (APCs). Macrophages play a tremendous role in clearing tagged antigens and releasing degradative enzymes to damage tissues and initiate healing.

Macrophages: trap, engulf and destroy pathogens (phagocytosis); present antigens for the adaptive immune response (antigen presentation); produce cytokines (including IL-12); and induce co-stimulatory molecules. Also, macrophages also bear CD14 (and LPS receptor), CD11b/CD18 complex (binds C3b and C4b, complement byproducts), scavenger receptor (binds sialic acid), TLR and F(c)R (described above). Please read about the precursor to macrophages, the monocyte.

Miscellaneous Cells

Important Classes

Immune system cells can be organized many different ways. The categories above are based primarily on origin. However, other broad classes are frequently used which are important to understand.

Hematopoiesis is the development of blood cells from hematopoietic stem cells (HSCs). A stem cell is any cell which can differentiate into other cell types; stem cells maintain their population via asymmetrical division — one daughter cell differentiates (the progenitor cell), and the other daughter cell remains in the bone marrow to continue the cycle. HSCs are sensitive to chemotherapy and radiation because of their frequent divisions; this sensitivity is the main barrier to tumor eradication. Stanford researcher Dr. Weissman and his colleagues developed stem cell enrichment to separate stem cells from bone marrow tissue.

The daughter cell that will differentiate is the progenitor cell, meaning it cannot self-renew and is committed to a particular cell lineage. There are two kinds of hematopoietic progenitor cells: lymphoid progenitor cells, which give rise to B cells, T cells and NK cells; and myeloid progenitor cells, which give rise to red blood cells (erythrocytes), various other white blood cells (neutrophils, eosinophils, basophils, monocytes, mast cells and dendritic cells) and platelet-generating cells called megakaryocytes.

Most hematopoiesis occurs in the bone marrow within a hematopoiesis inducing microenvironment (HIM) that is composed of cytokine-secreting stromal cells. Stromal cells include fat, endothelial, fibroblast and macrophage cells. Some of these cytokines operate via diffusion, while others are membrane-bound to the stromal cells and require direct cell-to-cell contact. Mostly identified via knockout mice, to the left are a few of the genes critical in hematopoiesis. These genes can impact many lineages or just one.

The lifespan of a cell can range from one 1 day for neutrophils to 120 days for erythrocytes to several decades for some T cells. With most blood cells dying due to aging, hematopoiesis produces just enough cells to keep up with cell death. The average human being produces 3.7E11 daily of white blood cells alone. To prevent overpopulation or misbehavior, cells undergo apoptosis (programmed cell death) or necrosis (death in response to cell injury). In apoptosis, the cell shrivels and then is phagocytosed by a macrophage. In necrosis, the cell swells, bursts and (in a crucial difference between apoptosis and necrosis) releases its contents into the surrounding area. This release can trigger an inflammatory response in surrounding tissues.

A component of the innate immune system, natural killer cells (NK cells) have genomic (not needed recombination, or RAG-independent) cell surface receptors which recognize classical Class I MHC molecules (and structural relatives like MICA, RAE-1 and H-60). Instead of directly recognizing pathogens, natural killer cells monitor cell surface molecules indicative of pathogenesis. This sensitivity allows natural killer cells to vigorously initiate natural killer cytotoxicity (by emptying granules of porforin and granzyme) and inflammation as soon as pathogenesis is detected, and is essential to protection against viruses and tumors.

Natural killer cells lack TcRs, CD4s and CD8; instead, they have: cell-surface activating receptors, which bind noncovalently to molecules with ITAMs; and on the cytoplasmic side, inhibitory receptors with ITIM(s) which — upon phosphorylation — recruit and activate SHP-1 & -2, which inhibit the activating receptors. The balance between activating signals and inhibitory signals is what determines whether a natural killer cell will destroy or bypass a microbe it encounters. There are many different inhibitory and activating receptors, but two well-studied ones are:

Macrophages (aka mononuclear phagocytes or mφs) have two main functions: phagocytosis and antigen presentation. In phagocytosis, the macrophage or monocyte cell-surface F(c) Receptor (FcR) binds to the antibody-antigen complex (an antibody bound to an antigen on the cell-surface of a pathogen). The pathogen, antigen and antibody are engulfed and degradative granules and enzymes break it down in the lysosome.

Digested antigen fragments are presented on the macrophage or monocyte cell-surface (in an MHC context) so other cells can recognize the antigen. Because macrophages present antigens, they are antigen presenting cells (APCs). Macrophages play a tremendous role in clearing tagged antigens and releasing degradative enzymes to damage tissues and initiate healing.

Since macrophages present antigens on their cell surface, they are known as antigen presenting cells (APCs). These antigens are then recognized by effector cells (B cells and T cells). Macrophages also activate cytokines, which stimulate differentiation and reproduction of lymphoid cells.

Macrophages are continuously maturing from circulating monocytes. Please read about the precursor to macrophages, the monocyte.

B cells (aka B lymphocytes) produce antibody when exposed to their complementary antigen. These antibodies can cause engulfment of infectious bacteria, neutralization of virions and induction of the complement cascade.

In the bone marrow, B cells complete their hematopoietic differentiation from stem cells into IgM⁺,IgD^weak immature virgin B cells. Next, in the medulla, B cells fully activate to become IgM⁺,IgD⁺ mature virgin B cells. Upon exposure to antigens, B cells in the medulla begin producing antibodies that flow through the lymph to the entire body. This helps create the body’s immune memory, with large amounts of antibody loaded in the bone marrow and at germinal centers. B cell activation occurs in the following steps:

1. An antigen flows through an afferent lymphatic vessel and into a node’s cortical sinus.
2. The antigen percolates through the node until getting trapped by reticular cells and dendritic cells.
3. Macrophages within the node trap and present the antigen. If it is a foreign antigen, either or both virgin B cells or memory B cells may react.

B-1 Cells

The B cells discussed so far are conventional B cells (aka B-2 cells). There is another subset of B cells known as B-1 cells (aka CD5 B cells, since some species’ B-1 cells display CD5). B-1 cells arise from stem cells during fetal life and self-renew via division of existing cells. While conventional B cells usually produce IgG, B-1 cells typically produce IgM and have little or no IgD. CD5 B cells secrete antibodies in response to TI-2 polysaccharides, leading to complement and removal of bacteria. This occurs within 48 hours of antigen exposure, as a bridge until the adaptive T cell response can activate. Unlike the T cell response, the B-1 response does not have memory. The table below (adapted from Immunology, 6^th edition) further compares and contrasts conventional (aka B-2) and B-1 cells.

After hematopoiesis in the bone marrow, granulocyte-monocyte progenitor cells differentiate into promonocytes. These promonocytes enter the bloodstream, where they mature further into monocytes; monocytes enlarge five- to ten-fold and become phagocytic while circulating for about up to three days in the blood. Next, these monocytes extravasate into tissues, where they become either fixed (tissue-specific) or free (wandering about the body) macrophages.

Monocytes move quickly to sites of infection in the tissues, and are one of the five major types of white blood cell. Monocytosis is the state of excess monocytes in the blood, and is indicative of disease. Monocytes can perform phagocytosis using intermediary (opsonising) proteins such as antibodies or complement that coat the pathogen, as well as by binding to the microbe directly via pattern-recognition receptors. Splenocytes are monocytes found in the spleen; macrophages are monocytes which have migrated from blood circulation to other tissues.

Instructional theories postulate that antigens play a central role in determining antibody specificity. Conversely, selective theories state that an antigen reacts with an already-existing antibody. Selective theories better explains acquired immune responses. Below is a history of antigen-antibody theories.

B cells were discovered approximately fifteen years after Burnett’s Clonal Selection Theory, proving that antibodies all pre-exist and that the repertoire is independent of the antigenic universe. This validated Burnett’s theory as well as Ehrlich’s similar (although much older) proposal. However, two key facets of Ehrlich’s theory were also disproven: B cells are exclusively dedicated to antibody production; and antibodies are not from food. The Clonal Expansion Theory is the modern explanation for antibodies and their origin.