Acquired/Adaptive Immunity

As the name implies, this type of immunity is not by birth rather acquired after previous antigenic exposures. Acquired immune responses are capable of selective elimination of pathogens.

5.3.1 Components of Adaptive Immunity

Adaptive immune responses are brought about by different classes of lymphocytes, namely B and T lymphocytes. The immune responses mediated through B lymphocytes are called antibody mediated or humoral immune response as B cells are capable of producing antibodies (or immunoglobulins) upon antigenic exposure, which bind with antigens and make them vulnerable for destructions. The cell-mediated immune responses are mediated through T lymphocytes that produce signals to activate phagocytic cells to destroy them.

T cells: They are so named due to their maturation in the thymus after derived from hematopoietic stem cells in bone marrow. They express antigen-binding receptors at their surface called T-cell receptor (TCR). The TCRs are able to recognize the correct antigen fragments after processed and presented through antigen-presenting cells (APC). Dendritic cells, macrophages, fibroblasts, and epi­thelial cells can act as APCs. These APCs express a surface protein called major histocompatibility complex (MHC) that recognizes self and nonself antigens.

Major histocompatibility complex (MHC): These are the cell surface proteins encoded by a group of genes present in chromosome 6 in humans.

B cells: B cells are matured in the bone marrow (or liver during fetal life), but in birds, the maturation of B lymphocytes takes place in the bursa of Fabricius, a lym­phoid organ found near the cloaca. B lymphocytes require antigenic exposure before final maturation after which they become immunocompetent. They have unique antigen-binding receptors and are able to interact directly with the antigens without the involvement of APCs. After interaction with foreign antigens through the receptors, B cells undergo proliferation and differentiations into plasma cells that are capable of producing antibodies. Plasma cells are short lived. Therefore, a portion of B cells are differentiated into long-lived memory B cells, which are able to respond quickly on re-exposure.

5.3.2 Lymphoid Organs

The organs at which lymphocytes are produced and matured are called lymphoid organs. There are two classes of lym­phoid organs.

Primary/central lymphoid organs: In primary lymphoid organs, the lymphocytes are produced and undergo matu­ration. The primary lymphoid organs are thymus, bone marrow, and bursa of Fabricius.

Secondary/peripheral lymphoid organs: In peripheral lym­phoid organs, the lymphocytes interact with antigens. They include lymph nodes, spleen, tonsils, and gut and mucosal associated lymphoid tissues.

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Recently, some abnormal lymph node-like structures were identified at the sites of chronic inflammation, cancers, and transplanted organs with graft rejection.

5.3.2.1 Primary Lymphoid Organs

Thymus: It is a bilobed structure situated above the heart. The lobes are encapsulated and are divided into lobules by connective tissue strands called trabeculae. The lobules are divided into outer cortex and inner medulla. There are stromal cells between the cortex and medulla composed of epithelium, dendritic cells, and macrophages. The thymic epithelial cells are also called nurse cells that surround the thymocytes in the cortex.

Thymus is mainly responsible for maturation of T cells. The progenitors of T cells derived from PHSC enter into the thymus as thymocytes and become immunoreactive and antigen-competent T cells. During the course of the devel­opment of thymocytes, they express antigen-binding receptors, and the T cells capable of recognizing foreign antigens and MHC molecule will be selected and released. The selection involves two steps. In the first step, there is positive selection of T cells that recognize self MHC. The T cells that are unable to recognize self MHC molecule will undergo apoptosis. In the second step (negative selec­tion), thymocytes with affinity receptor for self-antigen and self MHC are eliminated. So, ultimately the T cells that recognize both foreign antigen and MHC molecule are selected. This is called immune tolerance, and the thymus is called the organ of tolerance.

Bone marrow: It is a spongy tissue situated inside the bones. It is the primary hematopoietic organ. Initially, almost all the bones contain red bone marrow that creates blood cells. But, during the course of ageing, the marrow of long bones becomes fatty tissue and the hematopoiesis decreases. But the marrow of flat bones like ribs, sternum, and pelvis is active and hematopoiesis continues.

Bone marrow is the principal site for B-cell maturation. A portion of B cells enter into the secondary lymphoid organs to differentiate plasma cells upon antigenic stimu­lation. Other activated B cells become memory B cells or long-lived plasma cells reside in spleen and bone marrow. These are the persistent source of antibodies.

Bursa of Fabricius: It is the primary lymphoid organ of birds situated dorsal to the rectum and anterior to sacrum. It has a communication with cloaca through a short duct. The organ is composed of 12-20 longitudinal folds packed with numerous follicles separated through connective tis­sue layer. Each follicle contains B lymphocytes, dendritic cells, epithelium, and macrophages.

The bursa of Fabricius is the main site for antigen-committed B-cell maturation. Pre-bursal stem cells enter in the bursa around the seventh day of embryonic life and become bursal stem cells that undergo rapid differentiation in bursal microenvironment and become antigen-specific B cells and self-renewing post-bursal stem cells.

5.3.2.2 Secondary Lymphoid Organs

Lymph nodes: There are several lymph nodes strategically located in different anatomical locations to receive immu­nological signals from the body and provide an ideal microenvironment for immune cell communication. Each lymph node is divided into outer cortex that contains B cells. The inner medullary region contains both T and B cells. The paracortex between cortex and medulla contains T cells and dendritic cells. Both T cells and B cells enter the lymph nodes through endothelial venules and leave the node through efferent lymphatic vessels. T cells interact with dendritic cells during their movement, and their continual interactions facilitate to recognize foreign antigens entrapped in dendritic cells. Antigen-primed T cells then divide and induce immune reactions to eliminate it. Some of the dividing T cells also travel to B-cell-rich cortex and promote B-cell division and maturation to produce antibodies.

Spleen: It is one of the main secondary lymphoid organs in the left abdominal cavity beneath the diaphragm. It is the largest lymphatic organ of our body. Spleen is responsible for trapping of blood-borne antigens. Blood enters the spleen through splenic artery and leaves through splenic vein. The spleen is surrounded by a tissue capsule, which extends inward in the form of trabeculae to divide the spleen into two compartments, the red and white pulp. These red and white pulps are separated by marginal zone. The white pulp consists of mainly T lymphocytes surrounding splenic arteries and forms periarteriolar lym­phoid sheath (PALS). The red pulp consists of sinusoids filled with blood and populated by macrophages. The marginal zone is populated by lymphocytes and macrophages. The red pulp consists of venous sinuses filled with blood and cords of lymphatic cells, such as lymphocytes, erythrocytes, and macrophages. The defec­tive and old erythrocytes are destructed in the red pulp of spleen by the macrophages. When the antigens in the blood reach the spleen, they are trapped by dendritic cells situated in the marginal zone and carried to T-cell- rich PALS where the antigens are presented to TH cells with class II MHC molecules. Activated TH cells induce B-cell activation. Activated B cells and TH cells then migrate to primary follicles situated in the marginal zone. The primary follicles are differentiated into second­ary follicles upon antigenic exposure. The secondary follicles are like lymph nodes containing germinal centers populated by B cells and plasma cells surrounded by lymphocytes.

Mucosal associated lymphoid tissue (MALT): They are situated in the mucous membranes of the gastrointestinal, respiratory, and urogenital systems. They are populated with plasma cells. Due to the large surface area of the mucosal lining of the different systems of our body, the populations of plasma cells in the MALTs are far more than bone marrow, spleen, and lymph nodes together.

5.3.3 Antibody

The interaction of B cells with an antigen leads to prolifera­tion and differentiation of B cells to develop plasma cells. These plasma cells secrete antibodies specific to that particu­lar antigen that travels in the blood to neutralize the antigens.

Table 5.9 Different MALTs and their locations

Table 5.10 Serumlevelsof different immunoglobulins in different species

Source: Tizard (2013)

Therefore, antibodies are antigen-binding proteins produced by plasma cells. Antibodies are found in body fluids with maximum abundance in blood serum.

5.3.3.1 Structure of Antibody

Antibodies are glycoprotein in nature. They are mostly obtained from γ-globulin fractions of plasma protein and hence called immunoglobulins. They are heterodimer consisting of two identical heavy (H) and two identical light (L) molecular weight of 50,000 and 25,000, respectively. Each L chain is linked with H chain by a disulfide bond to form a heterodimer (H-L). There are also noncovalent interactions such as hydrogen bond and hydrophobic bonds between H and L chains. The other H and L chains are joined in a similar fashion to form another H-L heterodimer. These two identical H-L heterodimers are joined by a disulfide bond and non-covalent interactions to form a “Y”-shaped heterotetramer (H-L)² by a hinge region. The tip of the “Y,” the amino-terminal ends of both H and L chain containing 110-130 amino acids, varies greatly among different antibodies. This variable portion of the antibody is called V regions (V_L for L chain, V_H for H chain). The portion of V region which shows maximum variability among different antibodies is called complementarity-determining regions (CDRs) for both H and L chains. Like variable region, there is a constant (C) region at the tail of the “Y” for both L (C_L) and H (C_H) chains. Functionally, the immunoglobulin has two different regions, fragment antigen-binding (Fab frag­ment) and fragment-crystallizable region (Fc region) identified after digestion with the enzyme papain. The Fab region is a low-molecular-weight fraction (45,000 Da) having antigen-binding property. Another comparatively higher molecular weight fraction (50,000 Da) has no antigen­binding activity and is called Fc fragment (“fragment, crystallizable”).

5.3.3.2 Immunoglobulin Classes

There are five classes of immunoglobulins identified in mammals. They are immunoglobulin G (IgG), IgM, IgA, IgE, and IgD. Different classes of immunoglobulins are identified by their amino acid sequences in the constant region of the heavy chains. Normal serum levels of different immunoglobulins in different species have been presented in Table 5.10.

Immunoglobulin G (IgG): IgG has the smallest immuno­globulin molecule with highest abundance in the blood among other immunoglobulin classes. They are having molecular weight of about 180 kDa containing two identi­cal heavy chains (γ) and two different light chains (either κ or λ). IgG is produced from plasma cells of lymph nodes, spleen, and bone marrow. They can pass through capillary barriers and are thus found largely at the inflammatory sites when vascular permeability is increased. Based on their heavy-chain sequences, there are four IgG subclasses, namely IgG1, IgG2, IgG3, and IgG4. IgG1 has the highest concentration in the plasma, and the rest are numbered in accordance to their serum proportions, and IgG4 has the lowest concentration. Each IgG subclass also has different functional properties. IgG1, IgG3, and IgG4 are able to cross placental barriers easily and thus protect the fetus. IgG1 and IgG3 are primarily involved in opsonization due to their high affinity for Fc receptors, whereas IgG2 has the lowest affinity. The IgG3 is the most potent immunoglobulin for complement activation followed by IgG1. IgG4 is unable to activate complement system.

Immunoglobulin M (IgM): The IgM is secreted in the form of a pentamer consisting of five monomers each of 180 kDa; hence, the total molecular weight is about 900 kDa. They are primarily produced by plasma cells residing at secondary lymphoid organs. Their proportion in the serum is about 5-10% of the total immunoglobulins. Each IgM monomer consists of two light chains (κ or λ) and two identical heavy chains (μ). Five IgM monomers join together in circular fashion with the help of a small peptide chain of 15 kDa named J-chain. IgM has ten antigen-binding sites due to their pentameric structure. IgM is the first immunoglobulin produced in response to an infection, and it is also the first immunoglobulin synthesized in neonates. IgM is a potent complement activator compared to IgG and also helps in opsonization and viral neutralization, but due to their large size, they are found in very less concentration in the body fluids and rarely reach the site of inflammation.

Immunoglobulin A (IgA): They constitute around 10-15% of the total serum immunoglobulin and are mainly secreted from plasma cells located at the surfaces of the body such as skin, mammary gland, intestinal wall, and respiratory and urinary system. Thus, IgA is mostly found in the external secretions like milk, tears, saliva, and mucus. IgA is secreted as a dimer consisting of two single monomers having a molecular weight of 180 kDa, so secreted IgA has 360 kDa joined by J-chain. Each IgA monomer has two light chains and two heavy chains (α). IgA protects the mucous membrane of digestive, respiratory, and urinary systems against Salmonella, Vibrio cholerae, and Neisseria gonorrhoeae and polio, influenza, and reoviruses. As IgG is mostly secreted in milk, it protects the newborn from infec­tion during pre-weaning periods.

Immunoglobulin E (IgE): It was so named as it is induced by the E antigen of ragweed pollen. It has a molecular weight of about 190 kDa composed of two heavy chains (ε chain) and two light chains. Like IgA, it is also synthesized by the plasma cells under the skin, but in serum, it has extremely low concentration. IgE is mainly involved in allergic manifestation. It binds with Fc receptors of basophils and mast cells along with antigen and activates them. The activation of basophils and mast cells causes degranulation with the release of pharmacologically active substances that mediate allergic response. IgE is also involved in anti-parasitic defense. IgE has the shortest half-life (2-3 days).

Immunoglobulin D (IgD): It has a molecular weight of about 180 kDa and is composed of two δ heavy chains and two light chains. It is predominantly a membrane-bound immu­noglobulin expressed in mature B cells with a very little serum concentration (30 μg/mL). It constitutes about 0.2% of the total immunoglobin in serum. IgD induces basophils to release pro-inflammatory and antimicrobial mediators.

5.3.3.3 Immunoglobulin Variants

Isotypes: The isotypes of immunoglobulins are determined by the constant regions of the heavy chain, and the con­stant region determinants are called isotypic determinants. The variations in the constant region are due to variations in the genes that encode the heavy chains. Bovine IgG has three isotypes such as IgG1, IgG2, and IgG3 encoded by IGHG1, IGHG2, and IGHG3 genes, respectively. The different isotypes of immunoglobulins have different physical and functional properties. IgG2 is a potent agglu­tinin, whereas IgG1 does not have such a function. Subclasses of different immunoglobulins in different spe­cies have been presented in Table 5.11.

Allotypes: The allotypic immunoglobulin variants are generated due to allelic variations. In the previous section, we discussed bovine IgG isotypes like IgG1, IgG2, and IgG3 encoded by IGHG1, IGHG2, and IGHG3 genes, but there may be multiple alleles for IGHG gene which encode subtle amino acid differences and they are called allotypic determinants. IgA2 subclass has two allotypes designated as A2m(1) and A2m(2).

Idiotypes: The idiotypic immunoglobulin variants are resulted due to variations in the amino acid sequences in the variable regions of light and heavy chains.

Table 5.11 Subclasses of different immunoglobulins in different species

Source: Tizard (2013)

Immunoglobulin Superfamily These are a group of proteins having immunoglobulin-like domains containing 70-110 amino acids similar to variable and constant regions of immunoglobulin molecule. They share the common ances­tral primordial gene that encodes immunoglobulin domains. The proteins under the immunoglobulin superfamily are Ig-α/ Ig-β heterodimer of B-cell receptor, T-cell receptor, T-cell accessory proteins (CD2, CD4, CD8, and CD28), MHC class I and II molecules, cell adhesion molecule, and platelet- derived growth factors.

5.3.4 Recognition of Antigens by T and B Lymphocytes

B lymphocytes can recognize the epitopes that present on the surface of the pathogens or soluble factors released by the pathogens without the involvement of MHCs. The immunoglobulins present in the membrane of B lymphocytes specifically bind with the antigens. These membrane-bound immunoglobulins together with disulfide-linked

heterodimers called Ig-αΛlg-β are called B-cell receptor (BCR). Both Ig-α and Ig-β chains have long cytoplasmic tails consisting of 61 and 48 amino acids. The Ig-αZIg-β heterodimer is responsible for intracellular signaling after antigen binding.

In contrast to B cells, virus-infected host cells and cancer cells are recognized by T cells in conjugation with MHC. The antigen-binding sites on the T cells are called T-cell receptors (TCRs). TCRs are analogous to membrane-bound immunoglobins in B cells and also have both V and C regions, but TCR is unable to recognize antigen directly; rather, it recognizes short peptide fragments of antigens, which bind to MHC molecules through a process called antigen processing and presentation.

5.3.5 Antigen Processing and Presentation

The recognition of antigens by the T cells requires antigen processing and presentation by the APCs. In this process, foreign antigens are degraded into smaller peptides to interact with MHC molecules. There are two different mechanisms for exogenous and foreign antigens by which APCs process and present the antigens to T cells.

The exogenous antigens are engulfed by the APCs through endocytosis or phagocytosis, degraded by the endocytic processing pathways, and attached with class II MHC molecules. The antigen and MHC II complex then move to the surface of APCs and are recognized by T cells displaying CD4.

The endogenous antigens (viral proteins or cancer antigens) are produced with the cells. They are processed with the endoplasmic reticulum and subsequently attach with class I MHC molecules within the endoplasmic reticu­lum. The complexes are then transported to the cell mem­brane and recognized by T cells displaying CD8.

5.3.6 Monoclonal and Polyclonal Antibodies

Monoclonal antibodies bind to a single epitope, and poly­clonal antibodies are the heterogeneous immunoglobulin mixture against a single antigen and can bind with different epitopes of a single antigen. Monoclonal antibodies are pro­duced from the same clone of B cells, whereas polyclonal antibodies are produced from different B-cell clones. Mono­clonal antibodies have high specificity and reproducibility, but the production of monoclonal antibodies is time consum­ing and expensive. The polyclonal antibodies have high affinity, and cost of production is also less compared to monoclonal antibodies. Monoclonal antibodies are used in therapeutics, and polyclonal antibodies are used in research applications.

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