Information

12.3F: B-Lymphocytes (B-Cells) - Biology


Learning Objectives

Describe the overall function of B-lymphocytes and their activation by T-dependent antigens in terms of the following:

  1. the antigen receptor on their surface
  2. how they "process" exogenous antigens
  3. the type of MHC molecule to which they attach peptides
  4. the role of lysosomes in binding of peptides from exogenous antigens by MHC-II molecules.
  5. the type of cell to which they present peptides
  6. the types of cells into which activated B-lymphocytes differentiate

B-lymphocytes (B-cells) are responsible for the production of antibody molecules during adaptive immunity. B-lymphocytes refer to lymphocytes that are produced in the bone marrow and require bone marrow stromal cells and their cytokines for maturation. During its development, each B-lymphocyte becomes genetically programmed through a series of gene-splicing reactions to produce an antibody molecule with a unique specificity - a specific 3-dimensional shape capable of binding a specific epitope of an antigen (Figure (PageIndex{1})).

It is estimated that the human body has the ability to recognize 107 or more different epitopes and make up to 109 different antibodies, each with a unique specificity. In order to recognize this immense number of different epitopes, the body produces 107 or more distinct clones of B-lymphocytes, each with a unique B-cell receptor or BCR. In this variety of B-cell receptors there is bound to be at least one that has an epitope-binding site able to fit, at least to some degree, any antigen the immune system eventually encounters.

Typically, over 100,000 identical molecules of that unique antibody are placed on the surface of the B-lymphocyte where they can function as B-cell receptors capable of binding specific epitopes of a corresponding shape (Figure (PageIndex{2})). Naive B-lymphocytes can be activated by both T-dependent antigens and T-independent antigens.

Activation of naive B-lymphocytes by T-dependent antigens

In order for naive B-lymphocytes to proliferate, differentiate, and mount an antibody response against T-dependent antigens, such as most proteins, these B-lymphocytes must interact with effector T4-lymphocytes called TFH cells. All classes of antibody molecules can be made against T-dependent antigens and there is usually a memory response against such antigens.

B-Lymphocytes and T4-lymphocytes encounter antigens in secondary lymphoid organs such as the lymph nodes and the spleen. Using a lymph node as an example (Figure (PageIndex{3})A), soluble antigens, such as microbial polysaccharides and proteins and toxins, as well as microbes such as bacteria and viruses, enter the lymph node through afferent lymphatic vessels. By this time, complement pathway activation has coated these soluble antigens or microbes with opsonins such as C3b, which in turn can be degraded to C3d.

Located within the lymphoid tissues are specialized macrophages and specialized dendritic cells called follicular dendritic cells (FDCs). These macrophages have poor endocytic ability and produce few lysosomes. The FDCs are nonphagocytic. Both cell types, however, have complement receptors called CR1 and CR2 that bind to the C3b and C3d, enabling the antigens and microbes to stick to the surface of the macrophages and FDCs. However,because of the poor endocytic ability of the macrophages and the lack of endocytosis by the FDCs, the antigens and microbes are not engulfed but rather remain on the surface of the cells. In addition, the macrophages can transfer their bound antigens or microbes to FDCs (Figure (PageIndex{3})B).

Here the antigens and microbes in the lymph node can bind to complementary-shaped BCRs on naive B-lymphocytes directly, by way of macrophages, or via the FDCs (Figure (PageIndex{3})B).

Circulating naive B-lymphocytes, as a result of chemotaxis, enter lymph nodes through high endothelial venules. Any naive B-lymphocyte that bind antigens become activated and remain in the lymphoid nodes to proliferate and differentiate. Any B-lymphocytes not activated leave the lymphoid node through efferent lymphatic vessels and are returned to the bloodstream.

The first signal for the activation of a naive B-lymphocyte occurs when BCRs on the surface of the B-lymphocyte bind epitopes of antigens having a corresponding shape. A second signal is also needed for the activation of the naive B-lymphocyte. This is provided when the complement protein C3d on the microbial surface or soluble antigen binds to a complement receptor called CR2 on the surface of the naive B-lymphocyte.

Once bound, the antigen is engulfed, placed in a phagosome , and degraded with lysosomes. During this process, protein antigens are broken down into a series of peptide epitopes.These peptides eventually bind to grooves in MHC-II molecules that are then transported to the surface of the B-lymphocyte (Figure (PageIndex{4})).

Meanwhile, naïve T4-lymphocytes are being activated by epitopes of antigens bound to MHC-II molecules on antigen-presenting dendritic cells in the T-cell area of the lymph node and subsequently proliferate and differentiate into T4-effector lymphocytes such as TFH cells which remain in the lymph node. The T-cell receptors and CD4 molecules on TFH cells bind to the MHC-II molecules with bound peptide epitope on the B-lymphocyte. The binding of co-receptor molecules such as CD40L and CD28 on the surface of the effector T4-lymphocyte to the corresponding molecules CD40 and B7 on the surface of the B-lymphocyte further contribute to the interaction between these two cells (Figure (PageIndex{5})). This enables the TFH cells to produce cytokines such as interleukin-2 (IL-2) , interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-6 (IL-6) (Figure (PageIndex{5})).

Collectively these cytokines:

  1. Enable activated B-lymphocytes to proliferate.
  2. Stimulate activated B-lymphocytes to synthesize and secrete antibodies.
  3. Promote the differentiation of B-lymphocytes into antibody-secreting plasma cells. See Figure (PageIndex{6}).
  4. Enable antibody producing cells to switch the class or isotype of antibodies being produced.

YouTube animation illustrating production of antibodies by B-lymphocytes.

YouTube animation illustrating production of antibodies by B-lymphocytes against Streptococcus pyogenes.

Effector T4-lymphocytes also enable B-lymphocytes to undergo affinity maturation through a high rate of somatic mutation. This allows the B-lymphocytes to eventually "fine-tune" the shape of the antibody for better fit with the original epitope. After mutation, some antibodies fit better, some worse. To select for B-lymphocytes displaying antibodies with a better fit, the variant B-lymphocytes interact with cells called follicular dendritic cells (FDCs) in the germinal centers of the secondary lymphoid organs. The FDCs display the same antigens that activated the original B-lymphocyte. If the B-lymphocytes have high affinity antibodies for the antigen on the FDC, they are selected to survive. Those B-lymphocytes with low affinity antibodies undergo apoptosis.

With the exception of TFH cells which remain in the germinal centers of the lymph nodes and spleen, progeny of the activated B-lymphocytes and T4 effector lymphocytes leave the secondary lymphoid organs and migrate to tissues where they continue to respond to the invading antigen as long as it is present.

In the case of systemic infections or vaccinations where the antigens enter the bloodstream, plasma cells migrate to the bone marrow where antibodies can be produced for decades. After the antibodies are secreted by the plasma cells, they are found dissolved in the blood plasma and lymph. From here they can be delivered anywhere in the body via the circulatory system and the inflammatory response. In the case of infections of the mucous membranes, however, plasma cells only enter the mucous membranes where antibodies are only produced for a few months to a year or so.

During the proliferation and differentiation that follows lymphocyte activation, some of the B-lymphocytes stop replicating and become circulating, long-lived memory cells. Memory cells are capable of what is called anamnestic response or "memory", that is, they "remember" the original antigen. If that same antigen again enters the body while the B-memory cells (and T4-memory cells) are still present, these memory cells will initiate a rapid, heightened secondary response against that antigen (Figure (PageIndex{7})). This is why the body sometimes develops a permanent immunity after an infectious disease and is also the principle behind immunization.

Activation of B-lymphocytes by T-independent antigens

T-independent (TI) antigens are usually large carbohydrate and lipid molecules with multiple, repeating subunits. B-lymphocytes mount an antibody response to T-independent antigens without the requirement of interaction with effector T4-lymphocytes. Bacterial LPS from the Gram-negative cell wall and capsular polysaccharides are examples of TI antigens. The resulting antibody molecules are generally of the IgM isotype and do not give rise to a memory response. There are two basic types of T-independent antigens: TI-1 and TI-2.

a. TI-1 antigens arepathogen-associated molecular patterns or PAMPS such as lipopolysaccharide (LPS) from the outer membrane of the gram-negative cell wall and bacterial nucleic acid. These antigens activate B-lymphocytes by binding to their specific pattern-recognition receptors , in this case toll-like receptors, rather than to B-cell receptors (Figure (PageIndex{8})). Antibody molecules generated against TI-1 antigens are often called "natural antibodies" because they are always being made against bacteria present in the body.

b. TI-2 antigens, such as capsular polysaccharides, are molecules with multiple, repeating subunits. These repeating subunits activate B-lymphocytes by simultaneously cross-linking a number of B-cell receptors (Figure (PageIndex{9})).

For a Summary of Key Surface Molecules and Cellular Interactions of Naive B-Lymphocytes, see Figure (PageIndex{10}).

Summary

  1. B-lymphocytes are responsible for the production of antibody molecules during adaptive immunity.
  2. Antibodies are critical in removing extracellular microorganisms and toxins.
  3. B-lymphocytes refer to lymphocytes that are produced in the bone marrow and require bone marrow stromal cells and their cytokines for maturation.
  4. During its development, each B-lymphocyte becomes genetically programmed to produce an antibody molecule with a unique 3-dimensional shape capable of binding a specific epitope of an antigen, and puts molecules of that antibody on its surface that function as B-cell receptors or BCRs.
  5. Naive B-lymphocytes can be activated by both T-dependent antigens and T-independent antigens.
  6. In order for naive B-lymphocytes to proliferate, differentiate, and mount an antibody response against T-dependent antigens, such as most proteins, these B-lymphocytes must interact with effector T4-lymphocytes called TFH cells.
  7. The first signal for the activation of a naive B-lymphocyte occurs when BCRs on the surface of the B-lymphocyte bind epitopes of antigens having a corresponding shape.
  8. Once bound to the BCR, the antigen is engulfed, placed in a phagosome, and degraded with lysosomes. During this process, protein antigens are broken down into a series of peptide epitopes, bind to MHC-II molecules, and are transported to the surface of the B-lymphocyte.
  9. The T-cell receptors and CD4 molecules on TFH cells bind to the MHC-II molecules with bound peptide epitope on the B-lymphocyte which enables the TFH cells to produce cytokines that collectively enable the B-lymphocytes to proliferate, synthesize and secrete antibodies, differentiate into antibody-secreting plasma cells, and switch the class of antibodies being produced.
  10. By way of a mutation process called affinity maturation, activated B-lymphocytes are able over time to “fine-tune" the shape of the antibody for better fit with the original epitope.
  11. During the proliferation and differentiation that follows lymphocyte activation, some of the B-lymphocytes stop replicating and become circulating, long-lived memory cells that will initiate a rapid, heightened secondary response against that antigen if it again enters the body.
  12. T-independent (TI) antigens are usually large carbohydrate and lipid molecules with multiple, repeating subunits. B-lymphocytes mount an antibody response to T-independent antigens without the requirement of interaction with effector T4-lymphocytes, but the resulting antibody molecules are generally of the IgM isotype only and do not give rise to a memory response.

The five dimensions of B cell tolerance

B cell tolerance has been generally understood to be an acquired property of the immune system that governs antibody specificity in ways that avoid auto-toxicity. As useful as this understanding has proved, it fails to fully explain the existence of auto-reactive specificities in healthy individuals and contribution these may have to health. Mechanisms underlying B cell tolerance are considered to select a clonal repertoire that generates a collection of antibodies that do not bind self, ie tolerance operates more or less in three dimensions that largely spare autologous cells and antigens. Yet, most B lymphocytes in humans and probably in other vertebrates are auto-reactive and absence of these auto-reactive B cells is associated with disease. We suggest that auto-reactivity can be embodied by extending the concept of tolerance by two further dimensions, one of time and circumstance and one that allows healthy cells to actively resist injury. In this novel concept, macromolecular recognition by the B cell receptor leading to deletion, anergy, receptor editing or B cell activation is extended by taking account of the time of development of normal immune responses (4th dimension) and the accommodation (or tolerance) of normal cells to bound antibody, activation of complement, and interaction with inflammatory cells (fifth dimension). We discuss how these dimensions contribute to understanding B cell biology in health or disease.

Keywords: ABO incompatibility B lymphocytes accommodation antibodies tolerance.


Development

B cells are first produced in the fetal liver during the early stages of development. Based on studies involving mice, researchers noticed that during the production of these B cells, CXCL10 and CXCL12 (chemokine ligands) first attract CXCR3 and CXCR4-expressing pHSCs among other progenitors and influence their movement to the developing liver from the embryonic blood.

Here, they migrate into the stromal cells where they supply IL-7 among other molecules to the hematopoietic and B-lymphopoietic progenitors thereby activating the development of early B cells.

In adults, however, B cells are produced through the differentiation of hematopoietic stem cells located in the bone marrow (a primary lymphoid organ). From the primary lymphoid organ, they migrate to the secondary lymphoid organs where they undergo further development.

In the bone marrow (in the endosteal niche), the hematopoietic stem cells (HSCs), which are pluripotent in nature receive signals and thus undergo differentiation to produce progenitors known as lymphoid progenitor cells.

Unlike hematopoietic stem cells, these cells are multipotent and only capable of giving rise to a few types of cells. Once they receive the appropriate signal from the stromal cells in the bone marrow, these cells undergo division and differentiate to give rise to the earliest B cells.

Once the synthesis of RAG-1 and RAG-2 and Terminal deoxynucleotidyl transferase (TdT) synthesis in CD34+ lymphoid progenitors are activated by cytokines, these multipotent cells undergo D-J joining on the H chain chromosome which transforms them into pro B cells.

Further development is then characterized by the V segment joining the D-JH. When they express the membrane Mu (u) chains with surrogate light chains, these cells again change to become pre-B cells. Therefore, the pro/pre BI cells are some of the earliest types of B cells found in the bone marrow.

Generally, these cells are characterized by CD19 and CD117 expression as well as the arrangement of IgH chain loci in a D-J configuration.

During the development of B cells within the bone marrow, it's worth noting that antigens are not involved. Therefore, these cells are not yet exposed to antigens at this point - this phase is largely characterized by VDJ recombination.

The following are some of the characteristics of the different forms of B cells within the bone marrow:


Contents

T cell dependent mechanisms Edit

In a T-cell dependent development pathway, naïve follicular B cells are activated by antigen presenting follicular B helper T cells (TFH) during the initial infection, or primary immune response. [3] Naïve B cells circulate through follicles in secondary lymphoid organs (i.e. spleen and lymph nodes) where they can be activated by a floating foreign peptide brought in through the lymph or by antigen presented by antigen presenting cells (APCs) such as dendritic cells (DCs). [5] B cells may also be activated by binding foreign antigen in the periphery where they then move into the secondary lymphoid organs. [3] A signal transduced by the binding of the peptide to the B cell causes the cells to migrate to the edge of the follicle bordering the T cell area. [5]

The B cells internalize the foreign peptides, break them down, and express them on class II major histocompatibility complexes (MHCII), which are cell surface proteins. Within the secondary lymphoid organs, most of the B cells will enter B-cell follicles where a germinal center will form. Most B cells will eventually differentiate into plasma cells or memory B cells within the germinal center. [3] [6] The TFHs that express T cell receptors (TCRs) cognate to the peptide (i.e. specific for the peptide-MHCII complex) at the border of the B cell follicle and T-cell zone will bind to the MHCII ligand. The T cells will then express the CD40 ligand (CD40L) molecule and will begin to secrete cytokines which cause the B cells to proliferate and to undergo class switch recombination, a mutation in the B cell's genetic coding that changes their immunoglobulin type. [7] [8] Class switching allows memory B cells to secrete different types of antibodies in future immune responses. [3] The B cells then either differentiate into plasma cells, germinal center B cells, or memory B cells depending on the expressed transcription factors. The activated B cells that expressed the transcription factor Bcl-6 will enter B-cell follicles and undergo germinal center reactions. [7]

Once inside the germinal center, the B cells undergo proliferation, followed by mutation of the genetic coding region of their BCR, a process known as somatic hypermutation. [3] The mutations will either increase or decrease the affinity of the surface receptor for a particular antigen, a progression called affinity maturation. After acquiring these mutations, the receptors on the surface of the B cells (B cell receptors) are tested within the germinal center for their affinity to the current antigen. [9] B cell clones with mutations that have increased the affinity of their surface receptors receive survival signals via interactions with their cognate TFH cells. [2] [3] [10] The B cells that do not have high enough affinity to receive these survival signals, as well as B cells that are potentially auto-reactive, will be selected against and die through apoptosis. [6] These processes increase variability at the antigen binding sites such that every newly generated B cell has a unique receptor. [11]

After differentiation, memory B cells relocate to the periphery of the body where they will be more likely to encounter antigen in the event of a future exposure. [6] [2] [3] Many of the circulating B cells become concentrated in areas of the body that have a high likelihood of coming into contact with antigen, such as the Peyer's patch.

The process of differentiation into memory B cells within the germinal center is not yet fully understood. [3] Some researchers hypothesize that differentiation into memory B cells occurs randomly. [6] [4] Other hypotheses propose that the transcription factor NF-κB and the cytokine IL-24 are involved in the process of differentiation into memory B cells. [11] [3] An additional hypothesis states that the B cells with relatively lower affinity for antigen will become memory B cells, in contrast to B cells with relatively higher affinity that will become plasma cells.

T cell independent mechanisms Edit

Not all B cells present in the body have undergone somatic hypermutations. IgM+ memory B cells that have not undergone class switch recombination demonstrate that memory B cells can be produced independently of the germinal centers.

Upon infection with a pathogen, many B cells will differentiate into the plasma cells, also called effector B cells, which produce a first wave of protective antibodies and help clear infection. [6] [2] Plasma cells secrete antibodies specific for the pathogens but they cannot respond upon secondary exposure. A fraction of the B cells with BCRs cognate to the antigen differentiate into memory B cells that survive long-term in the body. [12] The memory B cells can maintain their BCR expression and will be able to respond quickly upon secondary exposure. [6]

The memory B cells produced during the primary immune response are specific to the antigen involved during the first exposure. In a secondary response, the memory B cells specific to the antigen or similar antigens will respond. [3] When memory B cells reencounter their specific antigen, they proliferate and differentiate into plasma cells, which then respond to and clear the antigen. [3] The memory B cells that do not differentiate into plasma cells at this point can reenter the germinal centers to undergo further class switching or somatic hypermutation for further affinity maturation. [3] Differentiation of memory B cells into plasma cells is far faster than differentiation by naïve B cells, which allows memory B cells to produce a more efficient secondary immune response. [4] The efficiency and accumulation of the memory B cell response is the foundation for vaccines and booster shots. [4] [3]

Memory B cells can survive for decades, which gives them the capacity to respond to multiple exposures to the same antigen. [3] The long-lasting survival is hypothesized to be a result of certain anti-apoptosis genes that are more highly expressed in memory B cells than other subsets of B cells. [6] Additionally, the memory B cell does not need to have continual interaction with the antigen nor with T cells in order to survive long-term. [4]

Memory B cells are typically distinguished by the cell surface marker CD27, although some subsets do not express CD27. Memory B cells that lack CD27 are generally associated with exhausted B cells or certain autoimmune conditions such as HIV, lupus, or rheumatoid arthritis. [2] [3]

Because B cells have typically undergone class switching, they can express a range of immunoglobulin molecules. Some specific attributes of particular immunoglobulin molecules are described below:

  • IgM: Memory B cells that express IgM can be found concentrated in the tonsils, Peyer's patch, and lymph nodes. [3] This subset of memory B cells is more likely to proliferate and reenter the germinal center during a secondary immune response. [4]
  • IgG: Memory B cells that express IgG typically differentiate into plasma cells. [4]
  • IgE: Memory B cells that express IgE are very rare in healthy individuals. This may occur because B cells that express IgE more frequently differentiate into plasma cells rather than memory B cells [4]
  • IgD only: Memory B cells that express IgD are very rare. B cells with only IgD are found concentrated in the tonsils. [13]

The receptor CCR6 is generally a marker of B cells that will eventually differentiate into MBCs. This receptor detects chemokines, which are chemical messengers that allow the B cell to move within the body. Memory B cells may have this receptor to allow them to move out of the germinal center and into the tissues where they have a higher probability of encountering antigen. [6]

Germinal center independent memory B cells Edit

This subset of cells differentiates from activated B cells into memory B cells before entering the germinal center. B cells that have a high level of interaction with TFH within the B cell follicle have a higher propensity of entering the germinal center. The B cells that develop into memory B cells independently from germinal centers likely experience CD40 and cytokine signaling from T cells. [14] Class switching can still occur prior to interaction with the germinal center, while somatic hypermutation only occurs after interaction with the germinal center. [14] The lack of somatic hypermutation is hypothesized to be beneficial a lower level of affinity maturation means that these memory B cells are less specialized to a specific antigen and may be able to recognize a wider range of antigens. [11] [15] [4]

T-independent memory B cells

T-independent memory B cells are a subset called B1 cells. These cells generally reside in the peritoneal cavity. When reintroduced to antigen, some of these B1 cells can differentiate into memory B cells without interacting with a T cell. [4] These B cells produce IgM antibodies to help clear infection. [16]

T-bet B cells are a subset that have been found to express the transcription factor T-bet. T-bet is associated with class switching. T-bet B cells are also thought to be important in immune responses against intracellular bacterial and viral infections. [17]

Vaccines are based on the notion of immunological memory. The preventative injection of a non-pathogenic antigen into the organism allows the body to generate a durable immunological memory. The injection of the antigen leads to an antibody response followed by the production of memory B cells. These memory B cells are promptly reactivated upon infection with the antigen and can effectively protect the organism from disease. [18]


How B-Cells Give Us Immunity

A young B-cell, called a naive B-cell, circulates in the bloodstream, usually ending up in the spleen or lymph nodes. It gets activated by an antigen, which can be any substance the body thinks is foreign, such as a piece of a virus, or a patch of a bacterium's cutter capsule. T-cells are often involved in this process.

The B-cell begins to transform into a plasma B-cell, whose specialized job it is to mass-produce the antibodies that match the activating invader—up to 10,000 antibodies per second.

Each plasma B-cell makes antibodies to only one antigen. They are very specific. Luckily, there are millions of them in our body so we can fight many different types of infection. Throughout the life of a B-cell, it makes these antibodies. They settle down mostly in the spleen and lymph nodes to pump out antibodies.

Some of the activated B-cells become memory B-cells, which have very long lives in the bone marrow, lymph nodes, and spleen. They remember the antigen they are specific for and are ready to respond quickly if they see it again. These are the cells that give us long-lasting immunity to different invaders.

When you get immunized, the vaccine contains antigens that stimulate the B-cells to produce antibodies that will then attack the virus, bacteria, or toxin you are being immunized against.   This mimics what is happening in your body when you are infected with that germ, but without the same risks of the disease caused by the germ or toxin.

Because B-cells have long memories, they can produce antibodies against germs and toxins for months and years, giving you a period of immunity.


CD20 as a gatekeeper of the resting stage of human B cells

CD20 is a B cell specific membrane protein and a target of therapeutic antibodies such as rituximab (RTX) 1 . In spite of the prominent usage of anti-CD20 antibodies in the clinic little is known about the biological function of CD20 2 . Here we show that CD20 controls the nanoscale organization of receptors on the surface of resting B lymphocytes. A CRISPR/Cas-based ablation of CD20 in Ramos B cells results in a relocalisation of the IgM B cell antigen receptor (IgM-BCR) and the co-receptor CD19. The resulting IgM-BCR/CD19 signaling synapse leads to transient B cell activation followed by plasma cell differentiation. Similarly to CD20-deficient Ramos cells, naïve human B cells treated with rituximab in vitro or isolated from patients during rituximab administration display hallmarks of transient activation characterized by the formation of the IgM-BCR/CD19 signaling synapse, followed by CD19 and IgM-BCR downregulation. Moreover, increased expression of specific plasma cell genes can be observed after rituximab treatment in relapsed CLL patients. In summary we identify CD20 as a gatekeeper of the resting state on human B cells and demonstrate that a disruption of the nanoscale organization of the B cell surface via CD20 deletion or anti-CD20 treatment profoundly alters B cell fate.


ACKNOWLEDGMENT

The authors would like to thank Dr Farid N. Faruqu and Prof Khuloud T. Al-Jamal, Institute of Pharmaceutical Science, Faculty of Life Sciences & Medicine, King's College London, for the use of their Nanosight. This work was funded by grants from British Heart Foundation and from the Rosetrees Trust. L.A.S. was funded by an ECR grant from the University of East London. The research was supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy's and St. Thomas' NHS Foundation Trust and King's College London. The views expressed are those of the author(s) and not necessarily those of the NHS, NIHR, or Department of Health and Social Care.


B cells and their function in the immune system

The protein Pdap1 (red) is located in the cytoplasm of B cells. Credit: Di Virgilio, MDC

Whenever a germ gets into the human body, the immune system usually responds immediately to fight off the enemy attacker. One of our defense system's most important strategies involves B lymphocytes, also known as B cells, which produce antibodies that target and neutralize pathogens. B cells play a central role in adaptive immunity and, together with T cells and components of the innate system, they protect the body against foreign pathogens, allergens and toxins.

A team led by Dr. Michela Di Virgilio, head of the Genome Diversification & Integrity Lab at the Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), has now identified a protein called Pdap1 that supports B cells in this important task while simultaneously protecting them from stress-induced cell death.

The lead authors of the study, which was published in the Journal of Experimental Medicine, are the two doctoral students—Verónica Delgado-Benito and Maria Berruezo-Llacuna—both members of Di Virgilio's lab. Researchers from the MDC's Berlin Institute of Medical Systems Biology (BIMSB) and the Experimental and Clinical Research Center (ECRC) were also involved. The ECRC is a joint institution of the MDC and Charité - Universitätsmedizin Berlin.

B cells must continuously adapt

"A successful humoral immune response, which is mediated by antibodies, is dependent on several factors," explains Di Virgilio. Mature B cells have to modify their genes (i.e., building instructions) in order to create antibodies that better match the distinguishing features on the surface of the invading pathogen. This is known as the lock-and-key principle and is achieved by somatic hypermutation, which mutates the pathogen-recognizing portion of the antibody molecule after the encounter and B cell activation.

Over the course of the humoral immune response, another part of the antibodies is transformed in a process known as class-switch recombination (CSR). Here, B cells change the isotype of the antibodies they produce. Instead of immunoglobulins of the isotype IgM, which are predominantly produced at the start of an infection, they may produce, for example, IgG antibodies, which have a different effector function. This process potentiates the ability of antibodies to effectively dispose of the pathogen.

The protein was found with the help of "gene scissors"

"In the beginning, we primarily wanted to understand how class switching works," says Delgado-Benito. "So we genetically modified a mouse B cell line using the CRISPR-Cas9 gene scissors to prevent them from producing certain proteins." In this way, she and the team discovered that without PDGFA associated protein 1 (Pdap1), less class switching occurs.

"In the next step, we generated mice where the gene for Pdap1 was switched off specifically in B cells," reports Berruezo-Llacuna. "This showed us that the protein is also crucial for somatic hypermutation." Without the protein, fewer such mutations occurred in the pathogen-recognizing part of the antibody, thus reducing the possibility to generate highly-specific variants.

B cells die more easily without Pdap1

"A particularly surprising finding to come out of our in vivo experiments, however, was that mouse B cells that are unable to produce Pdap1 die far more easily than is normally the case," adds Di Virgilio. Her team discovered that the protein protects B lymphocytes from stress-induced cell death. "Mature B cells experience cellular stressors particularly when they begin to grow and proliferate rapidly after contact with the pathogen," explains the researcher.

It seems that in unmodified animals, Pdap1 helps B cells to cope with this stress. Without the protein, however, a program is started that ultimately leads to cell death. "So Pdap1 not only helps the B lymphocytes to consistently produce the effective antibodies," says Di Virgilio. "It can also be seen as their protector."


B-cell milieu in NSCLC

B cells can exist in a continuum of naïve cells to terminally differentiated plasma cells within the TME and more specifically within the TLS [44]. Determining the ratio between these so-called “anti-tumour” TLS derived TIL-Bs and the “pro-tumour”, inhibitory Bregs is important to understand the biology and long-term outcome from this disease. This balance is likely influenced by the microenvironmental cues which play a role in determining B-cell polarity. CXCL13 and Lymphotoxin have been identified as two factors critical to the formation and development of lymphoid follicles in the gut [84], and in lung cancer, B cells produce CXCL13 and Lymphotoxin via TLR4 signalling which acts as a positive feedback loop to support the formation and high density of TLS [85, 86]. CXCR5 expressing B cells stimulated by CXC13 coupled CpG-ODN can trigger the cytolytic effect of CD8 + T cells leading to the abrogation of metastasis in 4T1.2 tumour-bearing mice [23]. Resveratrol, Lipoxin, Glucosides of Paeony have also inhibited Bregs through STAT3 and/or ERK inactivation leading to a reduction in IL-10 and TGF-β levels thus exerting an anti-tumour effect [87]. B-cell homeostasis and thus polarity will largely be determined by the degree of inflammation within the tumour, factors such as tissue hypoxia, intra-tumoural vascularity, cytokine milieu and cellular infiltration are all factors which are likely to exert control over the pro versus anti-tumour B-cell balance but as yet there is little evidence describing the Breg/B effector ratio in tumour biology, and this is likely due to the transient inducible nature of Bregs.


BIO 140 - Human Biology I - Textbook

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Chapter 26

The Adaptive Immune Response: B-lymphocytes and Antibodies

  • Explain how B cells mature and how B cell tolerance develops
  • Discuss how B cells are activated and differentiate into plasma cells
  • Describe the structure of the antibody classes and their functions

Antibodies were the first component of the adaptive immune response to be characterized by scientists working on the immune system. It was already known that individuals who survived a bacterial infection were immune to re-infection with the same pathogen. Early microbiologists took serum from an immune patient and mixed it with a fresh culture of the same type of bacteria, then observed the bacteria under a microscope. The bacteria became clumped in a process called agglutination. When a different bacterial species was used, the agglutination did not happen. Thus, there was something in the serum of immune individuals that could specifically bind to and agglutinate bacteria.

Scientists now know the cause of the agglutination is an antibody molecule, also called an immunoglobulin . What is an antibody? An antibody protein is essentially a secreted form of a B cell receptor. (In fact, surface immunoglobulin is another name for the B cell receptor.) Not surprisingly, the same genes encode both the secreted antibodies and the surface immunoglobulins. One minor difference in the way these proteins are synthesized distinguishes a naïve B cell with antibody on its surface from an antibody-secreting plasma cell with no antibodies on its surface. The antibodies of the plasma cell have the exact same antigen-binding site and specificity as their B cell precursors.

There are five different classes of antibody found in humans: IgM, IgD, IgG, IgA, and IgE. Each of these has specific functions in the immune response, so by learning about them, researchers can learn about the great variety of antibody functions critical to many adaptive immune responses.

B cells do not recognize antigen in the complex fashion of T cells. B cells can recognize native, unprocessed antigen and do not require the participation of MHC molecules and antigen-presenting cells.

B Cell Differentiation and Activation

B cells differentiate in the bone marrow. During the process of maturation, up to 100 trillion different clones of B cells are generated, which is similar to the diversity of antigen receptors seen in T cells.

B cell differentiation and the development of tolerance are not quite as well understood as it is in T cells. Central tolerance is the destruction or inactivation of B cells that recognize self-antigens in the bone marrow, and its role is critical and well established. In the process of clonal deletion , immature B cells that bind strongly to self-antigens expressed on tissues are signaled to commit suicide by apoptosis, removing them from the population. In the process of clonal anergy , however, B cells exposed to soluble antigen in the bone marrow are not physically deleted, but become unable to function.

Another mechanism called peripheral tolerance is a direct result of T cell tolerance. In peripheral tolerance , functional, mature B cells leave the bone marrow but have yet to be exposed to self-antigen. Most protein antigens require signals from helper T cells (Th2) to proceed to make antibody. When a B cell binds to a self-antigen but receives no signals from a nearby Th2 cell to produce antibody, the cell is signaled to undergo apoptosis and is destroyed. This is yet another example of the control that T cells have over the adaptive immune response.

After B cells are activated by their binding to antigen, they differentiate into plasma cells. Plasma cells often leave the secondary lymphoid organs, where the response is generated, and migrate back to the bone marrow, where the whole differentiation process started. After secreting antibodies for a specific period, they die, as most of their energy is devoted to making antibodies and not to maintaining themselves. Thus, plasma cells are said to be terminally differentiated.

The final B cell of interest is the memory B cell, which results from the clonal expansion of an activated B cell. Memory B cells function in a way similar to memory T cells. They lead to a stronger and faster secondary response when compared to the primary response, as illustrated below.

Antibody Structure

Antibodies are glycoproteins consisting of two types of polypeptide chains with attached carbohydrates. The heavy chain and the light chain are the two polypeptides that form the antibody. The main differences between the classes of antibodies are in the differences between their heavy chains, but as you shall see, the light chains have an important role, forming part of the antigen-binding site on the antibody molecules.

Four-chain Models of Antibody Structures

All antibody molecules have two identical heavy chains and two identical light chains. (Some antibodies contain multiple units of this four-chain structure.) The Fc region of the antibody is formed by the two heavy chains coming together, usually linked by disulfide bonds (Figure 1). The Fc portion of the antibody is important in that many effector cells of the immune system have Fc receptors. Cells having these receptors can then bind to antibody-coated pathogens, greatly increasing the specificity of the effector cells. At the other end of the molecule are two identical antigen-binding sites.

Figure 1: The typical four chain structure of a generic antibody (a) and the corresponding three-dimensional structure of the antibody IgG2 (b). (credit b: modification of work by Tim Vickers)

Five Classes of Antibodies and their Functions

In general, antibodies have two basic functions. They can act as the B cell antigen receptor or they can be secreted, circulate, and bind to a pathogen, often labeling it for identification by other forms of the immune response. Of the five antibody classes, notice that only two can function as the antigen receptor for naïve B cells: IgM and IgD (Figure 2 ). Mature B cells that leave the bone marrow express both IgM and IgD, but both antibodies have the same antigen specificity. Only IgM is secreted, however, and no other nonreceptor function for IgD has been discovered.

IgM consists of five four-chain structures (20 total chains with 10 identical antigen-binding sites) and is thus the largest of the antibody molecules. IgM is usually the first antibody made during a primary response. Its 10 antigen-binding sites and large shape allow it to bind well to many bacterial surfaces. It is excellent at binding complement proteins and activating the complement cascade, consistent with its role in promoting chemotaxis, opsonization, and cell lysis. Thus, it is a very effective antibody against bacteria at early stages of a primary antibody response. As the primary response proceeds, the antibody produced in a B cell can change to IgG, IgA, or IgE by the process known as class switching. Class switching is the change of one antibody class to another. While the class of antibody changes, the specificity and the antigen-binding sites do not. Thus, the antibodies made are still specific to the pathogen that stimulated the initial IgM response.

IgG is a major antibody of late primary responses and the main antibody of secondary responses in the blood. This is because class switching occurs during primary responses. IgG is a monomeric antibody that clears pathogens from the blood and can activate complement proteins (although not as well as IgM), taking advantage of its antibacterial activities. Furthermore, this class of antibody is the one that crosses the placenta to protect the developing fetus from disease exits the blood to the interstitial fluid to fight extracellular pathogens.

IgA exists in two forms, a four-chain monomer in the blood and an eight-chain structure, or dimer, in exocrine gland secretions of the mucous membranes, including mucus, saliva, and tears. Thus, dimeric IgA is the only antibody to leave the interior of the body to protect body surfaces. IgA is also of importance to newborns, because this antibody is present in mother&rsquos breast milk (colostrum), which serves to protect the infant from disease.

IgE is usually associated with allergies and anaphylaxis. It is present in the lowest concentration in the blood, because its Fc region binds strongly to an IgE-specific Fc receptor on the surfaces of mast cells. IgE makes mast cell degranulation very specific, such that if a person is allergic to peanuts, there will be peanut-specific IgE bound to his or her mast cells. In this person, eating peanuts will cause the mast cells to degranulate, sometimes causing severe allergic reactions, including anaphylaxis, a severe, systemic allergic response that can cause death.

Clonal Selection of B Cells

Clonal selection and expansion work much the same way in B cells as in T cells. Only B cells with appropriate antigen specificity are selected for and expanded (Figure 3). Eventually, the plasma cells secrete antibodies with antigenic specificity identical to those that were on the surfaces of the selected B cells. Notice in the figure that both plasma cells and memory B cells are generated simultaneously.

Figure 3: During a primary B cell immune response, both antibody-secreting plasma cells and memory B cells are produced. These memory cells lead to the differentiation of more plasma cells and memory B cells during secondary responses.

Primary versus Secondary B Cell Responses

Primary and secondary responses as they relate to T cells were discussed earlier. This section will look at these responses with B cells and antibody production. Because antibodies are easily obtained from blood samples, they are easy to follow and graph (Figure 4). As you will see from the figure, the primary response to an antigen (representing a pathogen) is delayed by several days. This is the time it takes for the B cell clones to expand and differentiate into plasma cells. The level of antibody produced is low, but it is sufficient for immune protection. The second time a person encounters the same antigen, there is no time delay, and the amount of antibody made is much higher. Thus, the secondary antibody response overwhelms the pathogens quickly and, in most situations, no symptoms are felt. When a different antigen is used, another primary response is made with its low antibody levels and time delay.

Figure 4: Antigen A is given once to generate a primary response and later to generate a secondary response. When a different antigen is given for the first time, a new primary response is made.

Active versus Passive Immunity

Immunity to pathogens, and the ability to control pathogen growth so that damage to the tissues of the body is limited, can be acquired by (1) the active development of an immune response in the infected individual or (2) the passive transfer of immune components from an immune individual to a nonimmune one. Both active and passive immunity have examples in the natural world and as part of medicine.

Active immunity is the resistance to pathogens acquired during an adaptive immune response within an individual ( Table ). Naturally acquired active immunity, the response to a pathogen, is the focus of this chapter. Artificially acquired active immunity involves the use of vaccines. A vaccine is a killed or weakened pathogen or its components that, when administered to a healthy individual, leads to the development of immunological memory (a weakened primary immune response) without causing much in the way of symptoms. Thus, with the use of vaccines, one can avoid the damage from disease that results from the first exposure to the pathogen, yet reap the benefits of protection from immunological memory. The advent of vaccines was one of the major medical advances of the twentieth century and led to the eradication of smallpox and the control of many infectious diseases, including polio, measles, and whooping cough.

Table 1: Active versus Passive Immunity

Natural Artificial
Active Adaptive immune response Vaccine response
Passive Trans-placental antibodies/breastfeeding Immune globulin injections

Passive immunity arises from the transfer of antibodies to an individual without requiring them to mount their own active immune response. Naturally acquired passive immunity is seen during fetal development. IgG is transferred from the maternal circulation to the fetus via the placenta, protecting the fetus from infection and protecting the newborn for the first few months of its life. As already stated, a newborn benefits from the IgA antibodies it obtains from milk during breastfeeding. The fetus and newborn thus benefit from the immunological memory of the mother to the pathogens to which she has been exposed. In medicine, artificially acquired passive immunity usually involves injections of immunoglobulins, taken from animals previously exposed to a specific pathogen. This treatment is a fast-acting method of temporarily protecting an individual who was possibly exposed to a pathogen. The downside to both types of passive immunity is the lack of the development of immunological memory. Once the antibodies are transferred, they are effective for only a limited time before they degrade.

T cell-dependent versus T cell-independent Antigens

As discussed previously, Th2 cells secrete cytokines that drive the production of antibodies in a B cell, responding to complex antigens such as those made by proteins. On the other hand, some antigens are T cell independent. A T cell-independent antigen usually is in the form of repeated carbohydrate moieties found on the cell walls of bacteria. Each antibody on the B cell surface has two binding sites, and the repeated nature of T cell-independent antigen leads to crosslinking of the surface antibodies on the B cell. The crosslinking is enough to activate it in the absence of T cell cytokines.

A T cell-dependent antigen , on the other hand, usually is not repeated to the same degree on the pathogen and thus does not crosslink surface antibody with the same efficiency. To elicit a response to such antigens, the B and T cells must come close together (Figure 5). The B cell must receive two signals to become activated. Its surface immunoglobulin must recognize native antigen. Some of this antigen is internalized, processed, and presented to the Th2 cells on a class II MHC molecule. The T cell then binds using its antigen receptor and is activated to secrete cytokines that diffuse to the B cell, finally activating it completely. Thus, the B cell receives signals from both its surface antibody and the T cell via its cytokines, and acts as a professional antigen-presenting cell in the process.

Figure 5: To elicit a response to a T cell-dependent antigen, the B and T cells must come close together. To become fully activated, the B cell must receive two signals from the native antigen and the T cell&rsquos cytokines.

Chapter Review

B cells, which develop within the bone marrow, are responsible for making five different classes of antibodies, each with its own functions. B cells have their own mechanisms for tolerance, but in peripheral tolerance, the B cells that leave the bone marrow remain inactive due to T cell tolerance. Some B cells do not need T cell cytokines to make antibody, and they bypass this need by the crosslinking of their surface immunoglobulin by repeated carbohydrate residues found in the cell walls of many bacterial species. Others require T cells to become activated.