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.

The T cell receptor (TCR) is a T cell surface receptor that recognizes antigens presented by MHC molecules. It is a heterodimer composed of either α and β chains or γ and δ chains, and which interacts with another T cell surface component CD3. αβ T cells are usually highly specific (adaptive immunity), while γδ T cells usually recognize broader antigen classes (innate immunity). TCR chains each contain two 60-75 amino acid domains: the amino-terminal variable (V) domain and the constant (C) domain. The V domain contains three hypervariable regions, similar to antibody complementarity determining regions.

The two chains are connected at a cysteine residue by a disulfide bond. The transmembrane region is 21 or 22 amino acids and positively charged, anchoring the TCR into the cell membrane and promoting TCR-CD3 interaction. Lastly, the cytoplasmic tail (carboxy-terminal end) is 5 to 12 amino acids. αβ and γδ TCR heterodimers resemble an Fab fragment attached to the cell membrane; due to this structural homology, they are included under the immunoglobulin superfamily.

αβ TCRs react only with peptide antigens. However, γδ TCRs can react to glycolipids and phospholipids (nonpeptides) presented by CD1. Furthermore, some γδ cells react with unprocessed peptide antigens not even presented by an MHC. Also, the most common γδ TCRs within a given species bind the most common pathogens that species encounters. These γδ TCRs directly binds microbes, and the γδ cells might directly kill pathogens.

Both innate and adaptive immunity rely heavily upon binding of the TCR to a peptide-MHC complex. γδ cells are characterized as part of innate immunity due to their rapid responses, low specificity and ability to bind free antigens. αβ cells, conversely, are clearly part of adaptive immunity. In addition to these functional differences, γδ cells and αβ cells are also structurally different. Allison, Garboczi and their coworkers found that a γδ receptor specific for non-presented microbial phospholipids has a deep binding cleft. Also, separate research found that γδ TCRs have a 111° bend between their V and C regions while αβ TCRs have a 147° bend.

T Cell Receptor Accessory Molecules

In B cells, membrane-bound antibodies associate with the Ig-α/Ig-β heterodimer to form the B cell antigen receptor. In T cells, the T cell receptor associates with its fellow membrane-bound component CD3 to form the TCR-CD3 membrane complex. Please note that CD3 is another component of the cell surface on T cells — TCR and CD3 are together on the T cell surface and interact together to transduce signals into the T cell. CD3 is essential for membrane expression of the TCR and for signal transduction. All CD3 chains contain a negatively charged amino acid (aspartic or glutamic acid) which interacts with one or two positively charged transmembrane TCR residues. Please note that B and T cell accessory molecules only participate in signal transduction after antigen interaction and do not actually influence antigen interaction.

Alone, the T cell receptor has low affinity for the peptide-MHC complex. For this reason, additional accessory molecules known as coreceptors or cell-adhesion molecules strengthen the bond between a T cell and a target or antigen-presenting cell. CD4 and CD8 are coreceptors which also distinguish cytotoxic T cells from helper T cells; CD4 binds Class II MHC molecules and CD8 binds Class I MHC molecules. Once bound, CD4 and CD8 also activate signal transduction. Additional coreceptors include CD2, LFA-1, CD28 and CD45R, all of which bind independently to other ligands on target or antigen-presenting cell. Once these coreceptors bind, the TCR scans for its appropriate peptide-MHC complex. Once TCR binds, membrane expression of cell-adhesion molecules is activated to tighten the intracellular bond. Cell-adhesion molecules play a role in tissue graft acceptance or rejection. If the grafted tissue bears MHC molecules which are similar to the host, then T cells will still respond to the MHC molecules.

T Cell Receptor Genomics

Genes encoding α, β, γ and δ chains are expressed only in T cells. Functional TCR genes are formed by rearrangements of V and J segments in α and γ genes, and of V, D and J segments (like IgH) in β and δ genes. The α, β, γ and δ genes also include C segments which do not rearrange. The C region contains a long exon encoding much of the C domain, followed by shorter exons encoding the connecting, transmembrane and cytoplasmic regions. In humans, α and δ gene segments are located on chromosome 14, while β and γ gene segments are located on chromosome 7. In mice, the α, β and γ gene segments are located on chromosomes 14, 6 and 13. In both mice and humans, δ gene segments are located between V and J α segments. The location of the δ gene segments is important: functional rearrangement of the α gene segments deletes C_δ, so that a T cell cannot co-express αβ TCRs with γδ TCRs. The table below describes human and murine TCR genes, and counts both pseudogenes (nonfunctional mutants) and functional genes.

Like the antibody L chain, the α chain is encoded by V, J and C segments. Like the antibody H chain, the β chain is encoded by V, D, J and C gene segments. Rearrangement of the TCR gene segments results in α VJ joining and β VDJ joining. After the rearranged TCR genes are transcribed, the α and β chains are expressed as a disulfide-linked heterodimer on the T cell membrane. Immunoglobulins can be bound or secreted, but TCRs are only membrane-bound. The TCR constant region, as shown in the figure above, includes constant (C), connecting, transmembrane and cytoplasmic sequences. Also, as opposed to the multiple different immunoglobulin C gene segments encoding different isotypes, α DNA has only one C segment and β DNA has duplicated J and C segments.

Pre-T cells expresses the recombination activating genes RAG-1 and RAG-2, as do pre-B cells. The RAG-1/2 recombinase recognizes conserved recombination signal sequences (RSSs) flanking each V, D and J gene segments in non-recombined TCR DNA. RAG-1/2 catalyzes V-J and V-D-J joining via the same deletions and inversions which occur in immunoglobulin genes. First, Rag-1/2 knicks one DNA strand between the coding and signal sequences and excises the resulting DNA loops. SCID mice (which lack B and T cells) are defective for a gene which repairs double-stranded DNA breaks; as a result, in SCID mice, the Ab and TCR D and J gene segments are not rejoined. RAG-1/2 only recombines TCR genes in T cells and Ig genes in B cells. This specificity is due to different recombinase regulatory systems, and uniquely configured chromatin in each lineage that allows recombinase to access only appropriate sites.

δ genes are located between the V and J segments of the &alpha gene. Thus, when the α gene recombines, the δ DNA is excised. In a phenomenon known as allelic exclusion, this mechanism prevents αβ TCRs and γδ TCRs from co-expressing. Another instance of allelic exclusion involves the two duplicate β gene J and C clusters. Having two duplicates means that at least one functional rearrangement is likely to take place. Once a functional rearrangement occurs, then rearrangement of the other cluster is inhibited. However, more than one α allele can undergo functional recombination; while this can lead to different α chains being expressed on the same cell surface, only one allele is MHC restricted and therefore functional. The mechanisms of TCR diversity are described below.

Isolating the T Cell Receptor

So Many Names For T Cells!

T cells — also known as T lymphocytes or thymocytes — are broken into two classes: CD8+ T cells, which are T cells which express CD8 cell surface proteins; and CD4+ T cells, which express CD4 cell surface proteins and are called TH cells. All T cells contain a T cell receptor (TCR), which is activated only by antigens embedded in an MHC complex. TcRs and antibodies are similar in that they both specifically bind antigens, but they are critically different because TcRs only bind MHC-associated antigens and an antibody will bind a free (floating) or membrane-bound antigen. When a TcR is activated by an antigen-MHC complex, its associated T cell will release a plethora of cytokines. Cytokines are used by the immune system to communicate within itself and with other tissues. To fully understand T cells, please read about T cell development upon completion of this article.

CD4 Cells    ·    TH Cells    ·    Helper T Cells

T cells expressing CD4 surface protein are called helper T cells (aka TH cells, T_H cells, effector T cells or any variation with the word ‘lymphocyte’ in place of ‘cell’. TH cells begin as TH₀ cells, but then differentiate into either TH1, TH2 or TH17 cells. TH1 and TH2 cells cross-regulate each other. However, an individual TH cell only produces one cytokine; thus, TH1 and TH2 effects are on the level of the entire body.

There are multiple diseases related to TH1 and TH2 cells. Experimental Allergenic Encephalomyelitis (EAE) is caused by a faulty TH1 response to myelin basic proteins of the central nervous system. Leprosey results from an inappropriate TH2 cell activity and is carried by a dominant allele. Allergies are causes by TH2 responses that lead to preferential IgE production. Also, as AIDS progresses, TH1 cells become TH2 cells, and TH17 cells rapidly disappear into the gut.

Virgin T cells migrate from the thymus through the blood, eventually flowing into capillaries at a lymph node and then through post-capillary venules (PCVs) into the node itself. T cells sometimes activate and exit lymph nodes to patrol the bloodstream. These circulating, activated T cells have two fates: extravasation to an infection site (detailed) below, following by reversion to a memory state and flow through the lymph to the nearest lymph node; or, if no extravasation occurs, reversion to a memory state after a few days of circulation, followed by crossing out of blood through PCVs and then flow to the nearest lymph node.

CD8 Cells    ·    TC Cells    ·    Cytotoxic T Cells

T cells expressing CD8 surface protein are called cytotoxic T cells (aka TC cells, T_C cells or any variation with the word ‘lymphocyte’ in place of ‘cell’). Cytotoxic T cells recognize surface markers on other cells in the body that label those cells for destruction. In this way, TCs help to keep virus-infected or malignant cells in check. Also, manipulation of antigen presenting TC cells is important for immune system targeting of tumor cells.

Traveling along chemical signals, thymocyte precursors migrate via blood from the bone marrow to the thymus. These cells have not yet rearranged their T cell receptor (TCR) genes and thus lack the T cell receptor (let alone CD3, CD4 or CD8); still lacking any characteristics of thymocytes, these immature T cells begin to divide furiously before individually undergoing four stages denoted double negative (DN) 1-4, named as such because the cells still lack CD4 and CD8 (CD4^-CD8^-). The four different DN steps — taking a total of ∼3 weeks — are described below, followed by the double-positive state (CD4⁺CD8⁺) and finally mature single-positive CD4⁺CD8^- or CD4^-CD8⁺ cells.

Thymic selection is a two-step process: positive selection, which induces apoptosis in thymocytes whose TCR cannot bind self MHC molecules; and negative selection, which induces apoptosis in thymocytes which bind self MHC molecules too well or in presence of a self peptide. Positive selection results in MHC restriction, and negative selection results in self-tolerance (meaning the thymocytes will not attack healthy self cells).

There are two proposed models as to how CD4⁺CD8⁺ cells mature into CD4⁺CD8^- or CD4^-CD8⁺ cells: the instructive model and the stochastic model. Neither model has been definitely proven nor disproven. The instructive model postulates that double-positive cells interact with either a Class I or a Class II MHC molecule, and are somehow signaled to differentiate into either CD4 or CD8 cells. The schotastic model postulates that repression of CD4 or CD8 is random and has nothing to do with TCR specificity. Only thymocytes whose TCR and coreceptor bind the same MHC molecule continue to mature.

The primary response is activation of naive thymocyte by a peptide-MHC complex. ∼48 hours after activation, the thymocyte enlarges into a blast cell and repeatedly divide to form a population of genetically identical cells (clonal expansion). Remember the G proteins described under transduction, and that G proteins help trigger the G₁ phase of the cell cycle). IL-2 concentration increases 100x in activated cells, helping induce up to 2-3 daily divisions for 4-5 days as well as thymocyte differentiation into either effector T cells or memory T cells.

T Cell Death

Over 98% of all thymocytes die during positive and negative selection, with the remaining cells entering the circulatory system to differentiate into effector or memory thymocytes. These T cells express two cell-surface proteins, Fas and Fas ligand (FasL), which are both essential for apoptosis via the Fas pathway. Upon activation, thymocytes increase Fas/FasL expression — the result is that over-stimulated cells are killed. This is essential for avoiding over-proliferation of thymocytes, and also for killing any self-reactive cells which avoided thymic selection.

One of the central mechanisms of the immune system is thymocyte activation, clonal expansion and differentiation (into either effector or memory cells). T cells are activated by binding of the TCR-CD3 complex to a processed antigen peptide bound to a Class I (CD8 cells, aka cytotoxic T cells) or a Class II (CD4, aka helper T cells) MHC molecule. A cascade of biochemical events is initiated, inducing the resting thymocyte to proliferate and differentiate. Induction occurs in two steps: initiation and signal generation, described below. This leads to expression of various gene products, listed below by how early they are expressed after initiation.

Naive thymocytes are those which have not yet encountered a peptide-MHC complex. Arrested in G₀, naive thymocytes have condensed chromatin, minimal cytoplasm and little transcriptional activity. They continuously recirculate through blood, lymph an lymph nodes. To activate, they need additional costimulatory signals to those described above. Signal 1 is the initial interaction between TCR-CD3 and peptide-MHC. Signal 2 is is provided by thymocyte CD28 and CD152 interaction with B7 proteins on the antigen-presenting cell. B7 proteins are constitutively expressed on dendritic cells, and in activated macrophages and activated B cells. B7 binds two proteins — CD28 and CD152 — which are both found on thymocyte membranes as disulfide-linked dimers. Binding of CD28 induces the cell to activate, while binding of CD152 represses activation. CD152 has a much higher B7 affinity than CD28, and its expression is activated by binding of CD28. Thus, CD152 is essentially a braking mechanism that maintains homeostasis; CD152 knockout mice have enlarged lymph nodes, enlarged spleen and die 3-4 weeks after birth. In the absence of a costimulatory signal, an unresponsive state called clonal anergy ensues (as opposed to clonal proliferation).

In essence, activation occurs when a dendritic cell simultaneously binds itself to a T_H’s antigen receptor (primary signal) and to its CD28 receptor (secondary signal). This signals to the dendritic cell that the antigen is foreign (dangerous) and that the next encountered cytotoxic thymocyte must be activated. Other times, dendritic cells are directly activated by an antigen via toll-like receptors and activate cytotoxic thymocytes — this is a critical example of how innate immunity activates adaptive immunity.

Before birth, the yolk sac, fetal liver and fetal bone marrow are the major sites of B cell maturation; after birth, B cells mature in the bone marrow. Many B cells are produced, but most die after a few weeks unless they encounter their specific antigen or nestle into a supportive lymphoid organ. An immature B cell bearing IgM in its membane leaves the bone marrow and matures to express both membrane-bound IgM and IgD (mIgM and mIgD) with a single antigenic specificity.

Naive B cells (have not encountered antigen) circulate in blood ad lymph and are carried to secondary lymphoid organs (notable the spleen and lymph nodes). If a B cell’s mIgM or mIgD interacts with its antigen, then the cell activates undergoes clonal expansion. This creates a population of genetically identical B cells (which express an identical antibody) that differentiates into memory B cells and plasma B cells. Also, some B cells undergo affinity maturation, whereby the average affinity of the antibodies they produce increases. Also, many B cells undergo class switching, whereby the B cells switch from producing µ isotype antibodies (IgMs) to produce γ, α or ε isotype antibodies.

When mature naïve B cell exit the bone marrow and begin recirculation, they are arrested in G₀ and typically die within a few weeks unless they are activated by their complementary antigen. An activated B cells undergoes proliferation and differentiation into memory and plasma cells, going from G₀ to G₁, S phase and then mitosis (cell division). There are two kinds of antigens, with each activating B cells along a unique pathway: thymus-dependent (TD) antigens and thymus-independent (TI) antigens. TD antigens requires direct contact with T_H cells (aka CD4 cells) and not just cytokines secreted by T_H cells. The humoral response to TI antigens is typically weaker than the TD response, does not form memory cells and predominantly leads to IgM secretion (indicating an absence of class switching). This is due to the critical role of T_H in affinity maturation, generating memory B cells and class switching to other isotypes.

The B cell response to thymus-independent antigens is split into two different pathways: the type-1 thymus-independent pathway (TI-1) is caused by lipopolysaccharide and other bacterial cell wall components; the type-2 thymus-independent pathway (TI-2) is caused by repetitive molecules such as bacterial flagellin and bacterial cell wall polysaccharides. Most TI-1 antigens are able to activate B cells regardless of their antigen specificity — they are polyclonal B cel activators or mitogens. TI-2 antigens activate B cells by binding mIg — however, cytokines secreted by T_H cells are required for full B cell proliferation and for class switching from IgM to other isotypes. The table below describes the TD, TI-1 and TI-2 antigens as well as their effects on the humoral response.

Activation By Thymus-Dependent Antigens

Two distinct signalling events are needed to push the resting naïve B cell into the cell cycle: signal 1 followed by signal 2. T_H cells are essential for activation of a B cell by thymus-dependent antigens. Binding of thymus-dependent antigens to a B cell’s mIg does not alone induce proliferation and differentiation without additional interaction with T_H membrane molecules as well as appropriate cytokines. The steps are described below:

Cross-linking of a membrane-bound immunoglobulin (mIg) with its complementary antigen initiates a signal transduction cascade that activates the attached B cell. Membrane-bound immunoglobulins have short cytoplasmic tails, rendering them unable to transduce activating signals on their own. However, each membrane-bound ligand-binding immunoglobulin associates with a single disulfide-linked signal-transducing heterodimer Ig-α/Ig-β to form the B cell receptor. Similarly, the pre-BCR consists of the Ig-α/Ig-β heterodimer associating with the surrogate light chain and µ heavy chains. Ig-α and Ig-β each contain a cytoplasmic tail with an 18-residue motif known as the immunoreceptor tyrosine-based activation motif (ITAM) which is also present in the T cell receptor (TCR). Also, just like the TCR, the BCR draws protein tyrosine kinases (PTKs) to its cytoplasmic tail upon cross-linking of the mIg by its complementary antigen.

Antigen-antibody crosslinking leads to phosphorylation of the tyrosines within the Ig-α and Ig-β ITAMs. This phosphorylation is performed by the receptor-associated PTKs Lyn, Blk and Fyn (similar to p56^Lck activity on TCRs). This ITAM phosphorylation creates docking sites for the critical proteins Syk (also a PTK, analogous to the TCR’s ZAP-70) and B cell linker protein (BLNK). These critical proteins provide docking sites for further proteins. Once BLNK has been phosphorylated by Syk, it recruits Bruton’s tyrosine kinase (Btk) and phospholipase Cγ2 (PLCγ2) so Syk can activate Btk, and so that Btk can then phosphorylate PLCγ2. Once PLCγ2 has been phosphorylated, it activates early calcium signaling and the initiation of pathways dependent on protein kinase C (PKC). The pathways activated by the BCR include small G protein pathways (for growth), PKC-dependent pathways and NF-κB production pathways — note the similarities to T cell activation.

In addition to the BCR, there are two membrane-bound components which provide stimulation (the B cell coreceptor) or inhibition (CD22). The B cell coreceptor is a complex of three proteins: CD19, which provides a long cytoplasmic tail with docking sites; CD21 (aka CDR2), which is a receptor for C3d; and CD81. C3d is a byproduct of complement that coats antigens — while the immunoglobulin binds the antigen, CD21 cross-links with C3d. This forms a BCR-antigen-BCcoR complex, allowing CD19’s cytoplasmic tail to interact with Ig-α and Ig-β and undergo phosphorylation. CD19’s phosphorylated cytoplasmic tail then binds signaling molecules, including the protein tyrosine kinase (PTK) Lyn, and hugely amplifies the activating signal. This explains how naïve B cells with low antigen affinity are still able to respond to low concentrations of antigen.

CD22 delivers a negative signal that makes activation of B cells more difficult. Activation of B cells leads to phosphorylation of the immunoreceptor tyrosine inhibitory motif (ITIM) in the cytoplasmic tail of CD22. Tyrosine phosphatase then binds the CD22 ITIM, stripping phosphates from the ITAMs of neighboring signaling complexes. Since ITAM phosphorylation is the core of B cell activation, phosphate removal deactivates the cell. CD22 knockout mice develop autoimmune diseases as they age, illuminating the importance of negative regulation.

Antibody production by activated B cells is the core the humoral response: antibody effects, such as complement activation by IgM and certain IgGs, opsonization via F(c)Rs and pathogen/toxin neutralization by high-affinity IgG and IgA; and processes related to B cell activation, such as T_H2 activation and cytokine production, germinal center formation, isotype switching, affinity maturation and memory cell production. The F(c) region of IgG binds to F(c) receptors, playing a critical role (along with receptors for complement byproducts) in clearing extracellular bacteria. Intracellular bacteria are cleared by cell-mediated immunity.

Antigens are grouped into thymus-dependent antigens and thymus-independent antigens. Activation by thymus-dependent antigens requires two signals: first, binding of the antigen itself to the B cell; second, binding of of a thymocyte to the B cell. Thymus-independent antigens, conversely, activate B cells on their own; in some cases, however, T_H cytokine secretion (but not binding) is needed for maximum B cell activity.

The thymus-dependent response requires linked recognition (aka associative recognition) whereby T_H and B cells must both recognize a given molecule as an antigen for it to activate the B cell. This was demonstrated in the cell-transfer experiment. Antigens in the blood are concentrated in the spleen, while antigens accessible by lymph are concentrated in the nearest lymph nodes and nodules. Lymph nodes trap more than 90% of all antigens which flow through them, whether those antigens are bound to free-floating antibodies or antibodies bound to antigen-transporting cells (such as Langerhans or dendritic cells) or macrophages.

T and B cell epitopes are not necessarily identical; for example, T cells respond well to internal viral proteins while B cells produce neutralizing antibodies to viral coat proteins. (Once virus-infected cells have been killed and unassembled virus proteins released, B cells specific for internal proteins can also be activated to make opsonizing antibodies to those proteins.) Attaching a carbohydrate to a protein can convert the carbohydrate into a T-dependent antigen; the carbohydrate-specific B cell internalizes the complex and presents peptides to Th2 cells, which in turn activate the B cell to make antibodies specific for the carbohydrate.