Activation, differentiation and memory of B and T cells

T-lymphocytes and B-lymphocytes are named after the organ in which they develop. T-lymphocytes do this in the thymus and B-lymphocytes in the bone marrow or liver during fetal development. Both T and B lymphocytes are derived from the same common lymphoid progenitor cells which, in turn, are derived from pluripotent hematopoietic stem cells, which give rise to all blood cell types including erythrocytes, leukocytes and platelets. In the case of T lymphocytes, they migrate to the thymus through the blood from hematopoietic tissues (these tissues are responsible for the production of blood cells). And B lymphocytes come from common lymphoid progenitor cells present in hematopoietic tissues.^1,2

Both cell types cannot be differentiated from a morphological point of view from each other, unless they have been previously activated by the antigen. When activated by the antigen, they proliferate and differentiate into effector cells. Effector B lymphocytes or plasma cells secrete antibodies. In contrast, effector T lymphocytes do not secrete antibodies, but a variety of proteins called cytokines act as local mediators.² 

B lymphocytes
B cells are the main effector cells of the humoral immune response (production of antibodies or immunoglobulins), as they are responsible for synthesising immunoglobulins against native antigens, i.e. not processed by an antigen-presenting cell. They are part of the adaptive immune system and their main functions are:^1,2

• Make antibodies against antigens.
• They act as antigen-presenting cells.
• Some become memory B cells after being activated by interaction with an antigen.

B lymphocytes have two processes of maturation and differentiation. One in the bone marrow and another in peripheral lymphatic tissues.

B lymphocytes act as antigen-presenting cells.
For a Th or cooperator lymphocyte to activate, it is necessary for a presenting cell to present antigen-derived peptides through MHC class II molecules. B lymphocytes participate in this process. The interaction with the antigen is carried out by the B lymphocytes through the antigen receptor (BCR) of their membrane, which is characteristic of these cells. The BCR has two structurally and functionally differentiated parts. The first is an Ig (immunoglobulin) molecule anchored in the plasma membrane, which differs only from (soluble) antibodies in a short sequence of transmembrane amino acids and cytoplasmic tails. The second is a heterodimer of two proteins – CD79alpha and beta- not covalently associated to Ig. In BCR, Ig is the component involved in binding with the antigen and CD79 proteins mediate the subsequent transduction of the signal into the cell nucleus.¹

When a B lymphocyte binds to the antigen through its BCR. In addition to receiving a first activation signal, it will internalize the antigen by endocytosis mediated by its receptor, which allows it to be processed in lysosomes and presented to the Th lymphocyte via class II MHC. Macrophages and dendritic cells can also present Th lymphocytes with those soluble antigens that can recognize their receptors, but since the B lymphocyte binds to the antigen via its specific Ig, it is capable of presenting antigens that are at concentrations between 100-1,000 times lower. Therefore, macrophages and dendritic cells can only present certain extracellular antigens effectively when they are at high concentrations, and usually B lymphocytes are the main cell presenting antigens to Th lymphocytes¹

But the BCR complex does not work alone. Other membrane proteins cooperate with it in the activation of the lymphocytes: they are the accessory molecules. These proteins also generate signals that are added to those of the BCR to activate the B lymphocyte when the specific signal is insufficient. Among them, the CD19/CD21/CD81 correceptor stands out, which coactivates B lymphocytes when the antigen is coated with complement (CD21 is a C3b receptor). Other accessory molecules (CD40, CD72, MHC-II) act during antigen presentation and interaction of B and T lymphocytes. B lymphocytes need these contacts and also soluble factors (cytokines) from T lymphocytes to synthesize antibodies.¹

The weak signal generated by the BCR complex and accessory molecules is amplified inside the cell by a set of biochemical reactions. These include phosphorylation/dephosphorylation of various intracellular substrates (lipids and proteins) by certain kinases or phosphatases, hydrolysis of membrane phospholipids, and increased calcium ions in the cytoplasm.The final result of the signals initiated by the BCR complex is the induction of certain groups of genes involved in the effector functions of the activated B lymphocyte.¹

Ripening of B lymphocytes in bone marrow.
The maturation or lymphocyte maturation of B lymphocytes occurs in the bone marrow from the same progenitor cell or multipotential stem cell that gives rise to other cells of the lymphoid lineage. In this organ takes place most of the maturation process of the B lymphocyte, which requires the participation of the stromal cells of the bone marrow (provides help via direct contact and via soluble factors) and involves the rearrangement of the genes of the Igs (immunoglobulins) and the sequential acquisition of different surface markers, which are modifying the phenotype of the lymphocyte. However, not all of the B lymphocytes generated go into the peripheral blood, but only those that are not potentially self-reactive leave the bone marrow for other lymphoid organs. The rest of the B lymphocytes are eliminated or inactivated by negative selection, which acts on immature B lymphocytes that recognize their own antigens in the medulla.¹

Most mature B cells (virgins) have IgM and IgD in their BCRs. A small part have other isotypes in whose synthesis they have specialized (IgG, IgA, IgE). B lymphocytes expressing CD5 synthesize IgM and are believed to be rapid and polyespecific response to bacteria.

Ripening and activation of B lymphocytes in peripheral lymphatic tissues.
B lymphocytes can be activated dependent on or independent of T lymphocytes:

1. T-lymphocyte-dependent activation.
   To generate antibodies it is not enough, in most cases, the participation of B lymphocytes alone, but the T lymphocytes play a determining role. The response of B lymphocytes to these T- or Timo-dependent antigens requires the direct contact of T and B lymphocytes. These T lymphocytes that help B lymphocytes in their activation are called cooperating T lymphocytes or Th, and provide help (once the antigen presented by the B lymphocyte itself is recognized through its BCR, which would be the first activation signal) through membrane molecules that bind to ligands present in B lymphocytes (as mentioned above) and through the release into the environment of soluble factors (cytokines) that bind to B lymphocyte receptors (the latter two processes would be the second activation signal).¹
2. Independent activation of T lymphocytes.
   There is a series of antigens that do not induce T-dependent type B response. Certain polymeric antigens, such as polysaccharides or bacterial lipopolysaccharides, can trigger a B response in the absence of T lymphocytes. They are called T- or Timo-independent antigens, i.e. they are unable to activate T lymphocytes¹

Formation of effector and memory B lymphocytes.
When B lymphocytes are activated, they proliferate generating germinal centers. Once the activated B lymphocytes receive the second activation signal from the Th lymphocyte, small pockets of B lymphocytes are formed and begin to proliferate. Some of the proliferating B lymphocytes move to the medullary cords of the lymph nodes and differentiate early into plasma cells that secrete IgM. This isotype is the most abundant Ig of the primary response, due to its early production. The rest of the B lymphocytes migrate to the primary follicles of the lymph nodes where they will divide, rapidly increasing the number of specific B lymphocytes. When the number of proliferating cells increases greatly, the morphology of the primary follicle changes to secondary follicle, in which there is a dense central zone, formed by a few clones of proliferating B lymphocytes, which is called the germinal center, and a less dense zone around with non-proliferating B lymphocytes, called the mantle. The lymphocytes that are proliferating in the germinal centre are called centroblasts and when they stop proliferating they become smaller, being called centrocytes.¹

In the course of activation and proliferation, the immune response improves continuously as B lymphocytes change the isotype of their immunoglobulin (Ig) and progressively increase their affinity for the antigen. This improvement of the humoral response (production of antibodies or immunoglobulins) is known as affinity maturation, and is a consequence of the processes of somatic hypermutation of the variable genes of the Igs and subsequent selection of the clones of B lymphocytes of greater affinity to the antigen. Th lymphocytes play an essential role, not only in the activation of B lymphocytes, but also in this process of selection of the most related B lymphocytes.¹

The first Igs that express virgin B cells in their membrane when they leave the bone marrow are IgM and IgD. But during the proliferation of centroblasts in the germinal center they can change isotypes and begin to synthesize IgG, IgA or IgE. This isotype change modifies the effect function of the antibodies (lysis, phagocytosis, inflammation…) but maintains the same antigenic specificity. The isotype change takes place in antigen activated B lymphocytes and is dependent on the cooperation of Th lymphocytes (Th1 and Th2). This process is performed by direct contacts between the B-lymphocyte membrane protein, CD40, and the Th-lymphocyte membrane protein, CD40L; and by soluble factors (cytokines). Certain cytokines favour selective switching to a particular isotype. Th lymphocytes therefore regulate both the production of antibodies by B lymphocytes (Th2) and their isotype (Th1 and Th2), which ultimately determines the effector function of that antibody.¹

B-lymphocytes that have undergone an isotype change may change back to another isotype, but they will never be able to express IgM and IgD again, as the loss of DNA during the change is irreversible. For this reason, IgM (majority isotype at the beginning of the primary response) is progressively replaced by other isotypes.¹

Finally, selected B cells (centers) that have proliferated and matured in germinal centers will differentiate into plasma cells or memory B cells.¹

• Plasma cells: Plasma cells do not divide, change isotypes, or undergo somatic mutation. In addition, they do not express neither Igs nor class II MHC on their membrane, making them independent of antigens and Th lymphocytes. They produce a large amount of the soluble form of their Ig. Unlike B-lymphocytes, plasma cells begin to process heavy chain RNA alternately. In this way, they stop synthesizing the membrane Ig and begin to secrete it externally in a soluble form. Because the secondary or peripheral lymphoid organs are very little vascularized, they migrate to the bone marrow where more than 90% of all Igs are produced. This type of cells die within a few days but the antibodies remain in the body for longer.¹
• Memory B lymphocytes: in the early stages of the immune response, when rapid production of antibodies is necessary to control the infection, most centers differentiate into plasma cells. When the infection has been controlled, the centers will differentiate into B-lymphocytes by memory. These lymphocytes re-circulate through the lymphoid organs and will be responsible for the secondary immune response. Because the primary response increases the number of memory B lymphocytes specific to a given pathogen (10-100 times more) and because they have already undergone affinity maturation and isotype change, the secondary immune response will be much faster and more efficient, and generally capable of killing the pathogen before it produces clinical symptoms. So memory B lymphocytes are responsible for the secondary immune response. They have a long life expectancy, even over 40 years.¹

Secondary immune response refers to when we come into contact a second or more times with a microbe. It is the memory B-lymphocytes, not the virgins, that are in charge of the response. The activation of virgin B lymphocytes, which do not have somatic hypermutation or affinity maturation, would be a useless expense. Therefore, the presence of specific antibodies against a certain antigen will inhibit the activation of virgin B lymphocytes. This inhibition occurs through the IgG receptor of B lymphocytes, called FCgammaRII. Pathogen-specific IgG will bind to the antigen via the BCR and FCgammaRII of virgin B lymphocytes by inhibiting them. However, in memory B lymphocytes, which also have the FCgammRII receptor, it produces activation and release of more specific IgG antibodies.¹

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T-lymphocytes
Unlike B lymphocytes, they cannot bind to native antigens because they need the help of antigen-presenting cells to process and present them. They are also part of the adaptive immune system and there are three main classes of T lymphocytes: cytotoxic T lymphocytes, collaborating T lymphocytes, and regulatory (suppressor) T lymphocytes. Cytotoxic T-lymphocytes directly kill infected cells while collaborating T-lymphocytes participate in the activation of macrophages, dendritic cells, B-lymphocytes and cytotoxic T-lymphocytes through the secretion of cytokines and through the expression on their surface of various co-stimulating proteins. Regulatory T lymphocytes or Treg would use similar strategies to inhibit the function of collaborating T lymphocytes, cytotoxic T lymphocytes, dendritic cells and B lymphocytes. Therefore, while B lymphocytes can act long-distance by secreting antibodies that enter the bloodstream and are widely distributed, T lymphocytes, although they can travel in the distance, only act locally on the nearest cells once there.²

Functional states of T lymphocytes.
T-lymphocytes can be found in the blood in three different functional states: virgin T-lymphocytes, memory T-lymphocytes and effector T-lymphocytes. Virgin T-lymphocytes are those cells that have not yet been in contact with the antigen since they left the thymus; memory T-lymphocytes are those that have been in contact with the antigen at least once, but have subsequently returned to the resting state prepared to respond again to the antigen against which they were stimulated. When the specific immune response is triggered, these virgin and memory T lymphocytes are activated, giving rise after several days to the effector T lymphocytes, which are characterized with the capacity to eliminate pathogens. Effector T lymphocytes are characterized phenotypically by having certain activation markers and, functionally, by being the cells that carry out the T-dependent functions of the specific or acquired immune response.¹

Activation and differentiation of T lymphocytes.
Activation of T lymphocytes begins in the secondary lymphoid organs. In these organs, antigens are processed and presented by MHC molecules (major histocompatibility complex) in antigen-presenting cells (including B-lymphocytes and dendritic cells). In addition to the signals mediated by the TCR/CD3 complex (a T cell receptor), other signals mediated by co-stimulatory molecules and/or cytokines are required for the proper activation of T lymphocytes. CD4 T cells recognise peptides presented in MHC class II molecules and CD8 T cells recognise peptides presented in MHC class I molecules.¹

Activation of CD8 T cells requires greater co-stimulation than CD4 T cells as they are potentially more dangerous. Such co-stimulation can occur in three different ways:

1. They can be directly activated by antigen-presenting cells (e.g. dendritic cells) with high levels of co-stimulating molecules (primarily CD80), inducing IL-2 synthesis and cytolytic function on that and other cells infected by the same virus.
2. When co-stimulation between these two cells is weak, CD8 T lymphocytes need the presence and collaboration of Th1s (we will talk about them later), who must recognize antigens of the same pathogen presented by the presenting cells. The union of the antigen-presenting cell (which was already attached to the CD8 T lymphocyte) and the Th1 lymphocyte can induce an increase in the co-stimulatory activity of the presenting cell, allowing the CD8 T lymphocyte to be activated.
3. Th1 lymphocytes activated by the antigen-presenting cell (which is also attached to the CD8 lymphocyte) can synthesize IL-2, which is capable of inducing activation of the CD8 T lymphocyte.

Once activated, CD4 T cells will become cytokine producing (Th) cooperating T cells and CD8 T cells will become cytotoxic (Tc) T cells. These (the CD8), on the other hand, will be in charge of the elimination of intracellular pathogens by cytolysis. CD4 T lymphocytes are called cooperators (Th, for helper) because they interact with other cell types (B lymphocytes, T lymphocytes, macrophages) and help them divide and develop their immune functions (e.g., synthesize antibodies, expand, destroy pathogens). This interaction involves membrane molecules of both cell types, but also soluble molecules, the cytokines, synthesized by the T lymphocyte, which bind to receptors of the other cell types. CD8 T cells are called cytotoxic or cytolytic (Tc) because they interact with other cell types (infected cells) that they lysate. There are two different mechanisms of lysis by cytotoxic T lymphocytes:¹

• Induction of programmed cell death (apoptosis) in target cells through specialized membrane molecules (CD95L/CD95).
• Destruction of the cell by formation of pores in the membrane (perforins) or by induction of apoptosis (granzymes).

Three subtypes can be distinguished between Th or T CD4+ lymphocytes: Th1, Th2 and Th17. Th1 and Th2 are not differentiated by the membrane molecules they express, but by the profile of cytokines they synthesize.¹

• The Th1 response seems to be triggered preferentially, against intracellular antigens (virus, bacteria); these responses are characterized by the high production of IL-2 and IFN-gamma, activating macrophages and NK or Tc lymphocytes. In addition, IFN-gamma induces isotype change and the production of opsonizing antibodies (IgG1 and IgG3) in B lymphocytes. Differentiation to Th1 cells is stimulated by IL-12 and IFN-gamma.¹
• The Th2 response appears to be triggered primarily against extracellular pathogens. Th2 lymphocytes are specialized in the activation of B lymphocytes for the secretion of antibodies (humoral immunity) with a majority production of IgG4 (very useful to neutralize viruses) and IgE that promotes the desgranulation of mast cells (involved in allergic reactions) and eosinophils (release mediators that destroy parasites). Differentiation to Th2 cells depends on IL-4 and secrete mainly IL-4, IL-5, IL-10 and IL-13.¹
• Th17 lymphocytes produce the proinflammatory cytokines IL-17 (IL17-A), IL-17F and IL-22. These cytokines induce the release of inflammatory mediators and chemokines by macrophages, fibroblasts and endothelial and epithelial cells, with consequent recruitment of neutrophils during immune responses directed against extracellular bacteria and fungi. On the other hand, Th17 cytokines appear to play an important role in the growth, differentiation and binding integrity of endothelial and epithelial cell surfaces.¹

Another population of CD4+ lymphocytes called regulatory T lymphocytes has been identified. Regulatory T lymphocytes or Treg regulate or suppress other cells of the immune system (mainly control effector T lymphocytes). They control immune responses to own or foreign antigens and help prevent autoimmune diseases. There are two main types: those produced in the thymus (“natural” tregs -nTreg or thymus tregs, tTreg) or those that differentiate from naïve (virgin) CD4 T cells in the periphery after antigen recognition in the presence of TGF-beta (“adaptive or induced” tregs – iTreg). Both Tregs cells express on their cell surface CD4+ the IL-2 receptor alpha chain (CD25) and the transcription factor FoxP3. Treg cells suppress the activation, proliferation and production of cytokines by CD4+ and CD8+ T cells and may also suppress B lymphocytes and dendritic cells. The nTreg perform this suppression by cellular contact with these cells and the iTreg through soluble messengers (without cellular contact) with immunosuppressive function, including TFG-β, IL-10 and adenosine.^1,2

All T lymphocytes need to be activated in order to develop their cooperative or cytolysis (effector) functions.

Pd: To understand the activation process of the T lymphocyte we recommend reading the post on the major histocompatibility complex (MHC). Click on this link.

Bibliography.

1. Regueiro González J.R., López Larrea C., González Rodriguez S. y Martínez Naves E. Inmunología: Biología y patología del sistema inmunitario. 4ª edición. Editorial Médica Panamerica, 2010.
2. Alberts Bruce, Johnson Alexander, Lewis Julian, Raff Martin, Roberts Keth y Walter Peter. Biología molecular de la célula. 5ª edición. Editorial Omega, 2010.