• Any substance such as proteins, carbohydrates and lipid present in bacteria, fungi, parasites and viruses are considered foreign to Human or any vertebrate host and have the capacity to induce immune response is known as Antigen.
• Lipids and nucleic acids are only antigenic when combined with proteins or polysaccharides
• Molecular weight of 10,000 Daltons or higher

• Immunogen:
  • Antigen which when introduced into vertebrate host, induces a specific immune response is called immunogen. They can mobilize immune system and provoke immune response
• Incomplete antigen:
  • A substance which can bind with specific antibody but cannot induce immune response by itself is known as incomplete antigen or hapten.
  • These small foreign molecules that is not antigenic and must be coupled with a carrier molecules to be antigenic. Once antibody are formed, they will recognize haptens.

• Antigenicity:
  • the ability of compound or foreign molecules to combine specifically with antibody is known as antigenicity.
• Immunogenicity:
  • the ability of the molecule to induce immune response. Immunogenicity of an antigen is determined by four properties; Foreigness, molecular size, chemical composition and heterogenicity and ability to be processed and presented on the surface of Antigen presenting cells (APCs).

Antigenic determinant

• Functional binding area which is immunologically active regions of an immunogen (or antigen) that binds to antigen-specific membrane receptors on lymphocytes or to Fab region of secreted antibodies. It is also called antigenic determinants or Epitopes.
• Antigenic determinant is the region of antigen molecule that is bound by Fab region of Antibody or TCR of T-lymphocytes
• The antibody or TCR does not interact with antigenic macromolecules as a whole but only with distinct portion of the molecule called antigenic determinant or epitope.
• Antigenic determinant may include- sugars, aminoacids and other organic molecules.
• A sequence of 4-6 amino acids is optimal size of an antigenic determinant of a protein antigen.
• Paratopes: the corresponding binding site of antibody (Fab region) to epitopes of antigen is called paratopes.
• Most antigens are multivalent. Note that the valancy of an antigen refers to the number of epitopes present in the antigen molecule. There are two type of epitopes in antigen. They are surface epitopes and hidden epitopes. Only surface epitopes can binds to Fab of antibody. Therefore, total number of surface epitopes represent functional valency of antigen. However, total valency of antigen is number of hidden and surface antigens.

 Properties of antigen/ factor determine immunogenicity

1. Foreignness:
   • In order to induce immune response molecule must be foreign to the host. Immune response (antibody) is not produced against self antigen
   • Immunogenicity of an antigen increase with increase in degree of foreignness. Generally greater the phylogentic gap between two species, greater will be the immune response.
   • For example, Bovine serum albumin (BSA) isolated from calf induce strong immune response when injected into Birds than in cattles.
   • However, there are some exception to above rule, for example- some hidden proteins of human beings ( Lens protein, sperm protein, thyroglobulin etc) when injected into same host, they induce immune response. Similarly collagen and cytochrome C are highly conserved during evolution, therefore these proteins from any species when injected do not induce or induce very little immune response.
2. Molecular size:
   • There is correlation between molecular size of antigens and immunogenicity.
   • Most active immunogen have molecular weight of 1 lakh (100,000 Da) or more.
   • Generally substances with molecular mass less than 5,000 Da are poorly immunogenic.
3. Chemical composition and heterogenicity:
   • Proteins are the most potent immunogen with carbohydrate ranking the second. In contrast Lipid and nucleic acids generally do not act as antigen (immunogen) unless they are complexed with protein or carbohydrates.
   • Homopolymers composed of polymer of single type of aminoacis or monosaccharides and are less immunogenic.
   • Heteropolymers are usually more immunogenic. Therefore from above it is clear that in order to be immunogenic, molecules must be complexed and heterogeneous in composition.
4. Susceptibility to antigen processing and presentation:
   • The development of both Antibody mediated immunity (AMI) and Cell mediated immunity (CMI) requires interaction of T-cells with antigen that has been processed and presented on the surface of Antigen presenting cells (APCs) along with MHC molecules.
   • Larger and insoluble molecules are more immunogenic than small and soluble molecule because larger molecules are more easily phagocytosed and presented by APCs.
   • The molecule that cannot be phagocytosed and presented by APCs with MHC are poorly immunogenic.
   • Furthermore the antigenic molecules must be digestable by hydrolytic enzyme of APCs. For example; polymer of D-aminoacids are poorly immunogenic because the hydrolytic enzymes of APC can only degrade L-aminoacids but not D-aminoacids.

Biological system that determines immunogenicity of antigen

Even if a molecule has properties to contribute to immunogenicity (such as foreignness, molecular size, chemical composition and heterogenicity and susceptible to Antigen processing and presenting), its ability to induce immune response depends on certain biological system.

1. Dose and route of administration:
   • Dose of antigen determines the amount of antibody produced in immunized host. An insufficient dose of antigen will not stimulate immune response either because it fails to activate enough T and B-lymphocytes or because of immunological tolerance in some cases. Conversely, excessive high dose can also induce tolerance.
   • Again single dose of most immunogen will not induce string immune response. Rather repeated administration over a period of weeks is required. Such repeated administration is called boosters.
   • If antigen is injected repeatedly, T-cells and B-cells are activated for long time and antibodies are produced in greater amount. Therefore, such principle is applied during vaccination.
   • Route of administration of antigen determine type of lymphoid organ where T cells and B-cells are activated for antibody production.
   • The immunogens are generally administrated through parenteral than orally.
   • The antigen administrated intravenously is carried to spleen where it activates T and B cells. On the other hand if antigen is injected subcutaneously, it enters into lymph node and activates T cells and B cells. 
2. Genotype of recipient host:
   • A major factor that determine immune response is genotype of recipient host. The genetic constitution of immunized host influence the type and degree of immune response.
   • For example, Hugh and McDeritt showed two strain of mice responded differently after exposure to synthetic immunogen. One strain produce high immune response while other strain produce low. When they were crossed, the F1 generation showed intermediate immune response.
   • The immune response of an animal to antigen is influenced and control by gene along with other environmental factors.
3. Adjuvants:
   • Adjuvants are substances that when mixed with antigen and injected with it, enhances the immunogenicity of that antigen.
   • Adjuvants are used in research and clinical trials to increase the immunogenicity of antigen with low immunogenicity or available in low amount. They are mixed with antigen to prepare vaccine.
   • For example; antibody response of mice toward BSA can be increased five folds or more if BSA is administered with adjuvants.
   • Some of known adjuvants are synthetic polysaccharides and bacterial lipopolysaccharides.
   • Adjuvants increase the immunogenicity by various ways. For examples- Alums
   • Alums is an adjuvant that prolongs the persistence of antigen. It is the only adjuvant approved to human use. When antigen is mixed with alum, it causes precipitation of antigen. When this precipitate Ag-alum complex is injected, it results in slow release of antigen from injection site and thus activates immune system for long time.
   • The alum precipitate also increase molecular size of antigen thus increase the susceptibility of antigen processing and presentation.
   • Other commonly used adjuvants are- LPS, heat killed Mycobacterial cells etc.
   • Effects of adjuvants:
     • Antigen persistence is prolonged
     • Co-stimulatory signals are enhanced
     • Local inflammation is increased
     • Non-specific proliferation of lymphocytes is stimulated

Types of Antigens

I. On the basis of immunogenicity:

1. Complete antigen or immunogen:

• Molecules that induce specific immune response by themselves are complete antigen or immunogen.
• Eg; Bacteriia, fungi, virus etc

2. Incomplete antigen or haptens:

• Antigens that cannot induce immune response by itself but can bind with specific antibody are called incomplete antigen or haptens.

3. Autoantigens:

• Some self-protein such as lens proteins, sperm protein, myolin basic protein, thyroglobulin, kidney protein and some heart muscle protein are sequestered from blood circulation throughout life. Therefore, these protein are recognized as foreign by T and B cells such that immune response is produced.

4. Allo-antigen or Iso-antigen:

• These antigens are individual specific antigen present in one individuals but not in other.
• Eg. Blood grouping antigen

5. Heterophilic or cross reacting antigen:

• If antigen produced against one antigen binds with other antigen then such antigen are called heterophilic or cross reacting antigen.
• Eg. Antibody produced against Rickettsia bind with some Proteus species. Similarly, antibody produced against M protein of Streptococcus pyogens cross reacts with heart muscle protein of human.

6. Super antigen:

• These antigen activates large fraction of t cells (upto 25%)
• Eg. Staphylococcus enterotoxins, shocks toxins, exfoliating toxin, Pyrogenic exotoxins

7. Tolerogen:

• Antigen that induce immunogenic tolerance ie. Unresponsiveness to antigen that is induced by prior exposure to that antigen.

8. Allorogen:

• Antigen that induce anaphylaxis (severe immediate hypersensitivity reaction) as a result of rapid generalized mast cell activation.

II. On the basis of need help of T cells:

1. Thymus dependent antigen:

• These antigen induce both AMI and CMI
• Eg. proteins

2. Thymus independent antigen:

• These antigen induce only AMI
• Eg. Polysaccharides

III. On the basis of origin:

1. Exogenous antigens:

• These antigen originate from outside and are foreign to host body
• These antigens enters to the body through inhalation, ingestion, or injection and start circulating in the body fluids and caught up by the APCs (Antigen processing cells such as macrophages, dendritic cells etc.).
• The engulfment of these exogenous antigens by APCs are mainly mediated by the phagocytosis. E.g. Bacteria, Fungi, Viruses etc.

2. Endogenous antigens:

• These antigens originate inside own body. These antigens are body’s own cells or sub fragments or compounds or the antigenic products that are produced as a result of normal cell metabolism, or because of viral or intracellular bacterial infection.
• The endogenous antigens are processed and presented by the macrophages to CD8 T cells (cytotoxic T – cells). E.g. Blood group antigens, HLA (Histocompatibility Leukocyte antigens) etc.

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• When specific lymphocytes confront antigens, there are two possibilities:
  • 1) The lymphocytes may be activated resulting to the immune responses.
  • 2) The lymphocytes may be inactivated or eliminated, leading to tolerance.
• The immune system employs many layers of protection in order to prevent the reaction of its cells and antibodies against host components which is termed as tolerance.
• Antigens inducing tolerance are termed as tolerogens or tolerogenic antigens rather than immunogens.
• On the basis of how and where the antigens are presented to the immune system, it can act as both the immunogen and a tolerogen.
• One of the fundamental properties of the normal immune system is self-tolerance.
• Self-tolerance is term given for the tolerance to self-antigens.
• In adults, most lymphocytes encounter with foreign antigen lead to an immune response aimed at elimination.
• But in case of foetus, due to the immature state of the immune system, exposure to antigens frequently leads to tolerance.

What are the factors promoting immunological tolerance?

• Regardless of foetal exposure, factors that promote immunological tolerance rather than stimulation of the immune system by a given antigen include the following:
  • High doses of antigen
  • Long-term persistence of antigen in the host
  • Intravenous or oral introduction
  • Absence of adjuvants (compounds that enhance the immune response to antigen)
  • Low levels of co-stimulation
  • Presentation of antigen by immature or in-activated antigen-presenting cells (APCs)

Importance of Immunological tolerance:

• Immunological tolerance plays significant role and is important for various reasons.
• In immunological tolerance, the lymphocytes that recognize self-antigens are either killed or inactivated, or their specificity is altered.
• This way, the individual becomes tolerant of self-antigens.
• Immunological tolerance plays role as therapeutic approach for the prevention of harmful immune responses.
• Also, induction of tolerance might also be helpful for the prevention of immune reactions to the products of newly expressed genes in gene therapy protocols.

Types and mechanisms of immunological tolerance:

• Tolerance is an immunologically specific phenomenon.
• When the specific lymphocytes recognize the antigens, it results in tolerance.
• The self-tolerance induction may occur either in immature self-reactive lymphocytes in primary lymphoid organs (central tolerance) or in mature lymphocytes in peripheral sites (peripheral tolerance).

Central tolerance:

• The first step for tolerance is the central tolerance.
• In central tolerance, if the T- or B-cell clones possess receptors that identifies self-antigens with high affinity, these cells are deleted before their maturation.
• It takes place in primary lymphoid organs (the bone marrow for B cells and thymus for T cells).
• However, central tolerance fails to account for unresponsiveness to antigens which are expressed only in peripheral tissues.
• Hence, peripheral tolerance mechanisms are induced for maintaining tolerance to such tissue-specific self-antigens.
• The occurrence of central tolerance takes place as the lymphocytes in course of maturation in the generative lymphoid organs, pass through a stage in which their encounter with antigen results in cell death or the expression of new antigen receptors or alteration in functional capabilities.
• Only self-antigens are present in the thymus and bone marrow.
• It is because the foreign antigens are not transported to the thymus.
•  Instead, it is captured and migrated to peripheral lymphoid organs, such as the lymph nodes, spleen, and mucosal lymphoid tissues.
• Thus, the developing lymphocytes normally interacts only with self-antigens in the primary lymphoid organs.
• This encounter of immature lymphocytes with self-antigens has several possible outcomes:
  • Cells may die by apoptosis
  • Undergo deletion
  • In case of B cells, it changes its specificity and in case of T cells, it develops into regulatory tolerance

Peripheral tolerance:

• The occurrence of peripheral tolerance takes place when the mature lymphocytes that recognize self-antigens loses its ability to respond to that antigen, or lose their viability and become short-lived cells, or are induced to die by apoptosis.
• The importance of peripheral tolerance is listed as:
  • To maintain unresponsiveness to self-antigens that are expressed in peripheral tissues and not in primary lymphoid organs.
  • For the tolerance to self-antigens that are expressed in adult life after the production of mature lymphocytes.
• The mature lymphocytes repertoire consists of cells able to recognize such self-antigens.
• The responses of the mature lymphocytes to these antigens are strictly regulated to maintain self-tolerance.
• However, it is still unclear which self-antigens induce central or peripheral tolerance.
• The basic principle is that the choice between lymphocyte activation and tolerance is determined by the properties of the antigens, by the state of maturation of the antigen-specific lymphocytes, and by the types of stimuli received when these lymphocytes encounter self-antigens.

What is Antigen sequestration?

• Along with the mechanisms of central and peripheral tolerance, an effective way to avoid self-reactivity is sequestration (compartmentation) of antigens.
• For instance, the anterior chamber and lens of the eye are regarded as sequestered sites, without lymphatic drainage.
•  It also possess tissue-specific privileged antigens that are generally kept apart from interaction with immune cells.
• This sequestration enables these antigens to avoid encounter with reactive lymphocytes under normal circumstances; if the antigen is not exposed to immune cells, there is little possibility of reactivity.
• However, one possible outcome of sequestration is that the antigen is never encountered by developing lymphocytes, and thus active tolerance to the sequestered antigen is not established.
• If barriers between immune cells and the sequestered antigens are ruptured (for example by trauma), the newly released antigen may be taken as foreign as it was not previously encountered.
• Trauma to one eye that permits entry of immune cells can lead to inflammation in that eye, causing tissue destruction and impaired vision.
• In these cases, the other eye may also become inflamed due to the sudden entry of clones of these recently activated immune cells identifying newly discovered tissue-specific antigens.
• This is not to convince that a lack of exposure to the immune system is the only factor that mediates immune privilege.
•  A locally immunosuppressive microenvironment in tissues is traditionally considered immune privileged, such as the eye and central nervous system (CNS).
• In addition to other active mechanisms, it is believed to bias the immune response toward tolerance in these locations.

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• Antigen processing is a metabolic process that digests the proteins into peptides which can be displayed on the cell membrane together with a class-I or class-II MHC molecules and recognized by T-cells.
• Antigen presentation is the process by which certain cell in the body especially antigen presenting cells (APCs) express processed antigen on their cell surface along with MHC molecules in the form recognizable to T cell.
• If antigen is presented along with class-I MHC molecule, it is recognized by CD8⁺ Tc-cell and if presented along with class-II MHC molecule, it is recognized by CD4⁺ TH cells.

On the basis of types of antigen to be processed and presented, antigen processing and presenting pathway are of two types:

Cytosolic pathway of antigen processing and presentation

• Cytosolic pathway processed and presented the endogenous antigens i.e. those generated within cell eg. Viral infected cells, tumor cells and intracellular pathogens (M. tuberculosis, Histoplasma capsulatum).
• The processed antigen is presented on the cell membrane with MHC-class I molecule which is recognized by CD8⁺ Tc-cell for degradation.

Steps involved in cytosolic pathways are:

• Proteolytic degradation of Ag (protein) into peptides
• Transportation of peptides from cytosol to RER
• Assembly of peptides with class I MHC molecules

i. Proteolytic degradation of proteins into peptides:

• Intracellular proteineous antigen are larger in size to be bound to MHC molecule.
• So, it is degraded into short peptides of about 8-10 amino acids.
• These proteins are degraded by cytosolic proteolytic system present in cell called proteasome.
• The large (20S) proteasome is composed of 14 sub-units arranged in barrel-like structure of symmetrical rings.
• Some, but not all the sub-units have protease activity.
• Proteins enter the proteasome through narrow channel at each end.
• Many proteins targeted for proteolysis have a small protein called ubiquitin attached to them.
• Ubiquitin attached to them ubiquitin-protein complex consisting of 20S proteasome and 19S regulatory component added to it.
• The resulting 26S proteasome cleaves peptide bonds which is ATP-dependent process.
• Degradation of ubiquitin protein complex is thought to occur within the central hollow of the proteasome to release peptides.

ii. Transportation of peptides from cytosol to Rough Endoplasmic Reticulum (RER):

• Peptides generated in cytosol by proteasome are transported by TAP (transporter associated with antigen processing) into RER (Rough endoplasmic reticulum) by a process which require hydrolysis of ATP.
• TAP is membrane spanning heterodimer consisting of two proteins, TAP1 and TAP2.
• TAP has affinity for peptides having 8-16 amino acids.
• The optimal peptide length required by class-I MHC for binding is nine, which is achieved by trimming the peptides with the help of amino-peptidase present in RER. Eg. ERAP.
• In addition to it, TAP favor peptides with hydrophobic or basic carboxyl terminal amino acids, that preferred anchor residues for class-I MHC molecules.
• TAP deficiency can lead to a disease syndrome that has both immune-deficiency and auto-immunity aspects.

iii. Assembly of peptides with class-I MHC molecule:

• Like other proteins, the α-chain and β₂ microglobulin components of the class-I MHC molecule are synthesized on polysome along the rough endoplasmic reticulum.
• Assembly of these components into stable class-I MHC molecule that can exit the RRE require binding of peptides into peptide binding groove of class-I MHC molecules.
• The assembly process involves several steps and needs help of molecular chaperone.
• The first molecular chaperone involved in assembly of class-I MHC is calnexin.
• It is a resident membrane protein of RER.
• Calnexin associated with free class-I α-chain and promotes its folding.
• When β₂-microglobulin binds class-I α-chain, calnexin is released and class-I MHC associates with another chaperone calreticulin and tapasin (TAP-associated protein).
• Tapasin brings TAP transporter carrying peptides to the proximity with class-I MHC molecule and allows to acquire the antigenic peptides.
• An additional protein with enzymatic activity, ERp57, form disulfide bond to tapasin and non-covalently associates with calreticulin to stabilize the interaction and allows release of MHC-I-class after acquiring antigenic peptides.
• As a consequence, the productive peptide binding with MHC of class-I releases from the complex of calreticulin, tapasin and ERp57, exit from RER and displays on the cell surface via golgi complex.

Endocytic pathway of antigen processing and presentation:

• The endocytic pathway processed and present the exogenous Ag. i.e. antigens generated outside the cells. E.g. Bacteria.
• At first APC phagocytosed, endocytosed or both, the antigen.
• Macrophage and dendritic cells internalize the antigen by both the process.
• While other APCs are non-phagocytic or poorly phagocytic. E.g. B cell internalize the antigen by receptor mediated endocytosis.
• Then antigen is processed and presented on the cell surface along with class-II MHC molecules which are recognized by CD4⁺ TH cell.

Steps involved in endocytic pathway:

• Peptide generation from internalized molecules (Ag) in endocytic vesicles.
• Transport of class-II MHC molecule to endocytic vesicles.
• Assembly of peptides with Class-II MHC molecules.

i. Peptide generation from internalized molecules (Ag) in endocytic vesicles:

• Once an antigen is internalized, it is degraded into peptides within compartments of endocytic processing pathway.
• The endocytic pathway appears to involve three increasingly acidic compartments, early endosomes (pH 6-6.5), late endosomes or endo-lysosome (pH 5-6) and lysosomes (pH 4.5-5).
• The internalized antigens move from early to late endosomes and finally to lysosomes, encountering hydrolytic enzymes and a lower pH in each compartment.
• Within the compartment, antigen is degraded into oligopeptides of about 13-18 residues.
• The mechanism by which internalized Ag moves from one endocytic compartment to next has not been clearly demonstrated.
• It has been suggested that early endosome move from periphery to inward to become late endosome and finally lysosomes.
• Alternatively, small transport vesicles may carry Ag from one compartment to next.

ii. Transport of class-II MHC molecule to endocytic vesicles:

• When class-II MHC molecules are synthesized within RER, three pairs of class-II αβ-chains associated with a pre-assembled trimer of a protein called invariant chain (Li, CD74).
• This trimeric protein prevents any endogenously antigen to bind to the cleft.
• The invariant chain consists of sorting signals in its cytoplasmic tail.
• It directs the transport of class-II MHC molecule to endocytic compartments from the trans-golgi network.

iii. Assembly of peptides with class-II MHC molecules:

• Class-II MHC-invariant chain complexes are transported from RER through golgi complex and golgi-network and through endocytic compartment, moving from early endosome to late endosome and finally to lysosome.
• The proteolytic activities increase in each compartment, so the invariant is slowly degraded.
• However, a short fragment of invariant chain remained termed as CLIP (Class-II associated invariant chain).
• CLIP physically occupies the peptide binding, cleft of class-II MHC molecule, presumably preventing any premature binding of antigenic peptides.
• A non-classical class-II MHC molecule known as HLA-DM is required to catalyze the exchange of CLIP with antigenic peptides.
• The reaction between HLA-DO, which binds to HLA-DM and lessens the efficiency of the exchange reactions.
• Conditions of higher acidity in endocytic compartment weakens the association of DM/DO and increase the possibility of antigenic peptide binding despite of DO.
• As with class-I MHC molecule, peptide binding is required to maintain the structure and stability of class-II MHC molecules.
• Once a peptide has bound the peptide-class II MHC complex is transported to the plasma membrane where neutral pH enables the complex to assume the compact and stable form.

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• The antibody mediated immune response is also known as humoral immunity.
• It is the host defense mechanism that are mediated by antibody present in plasma, lymph and tissue fluids.
• It is the type of adaptive immune system responsible for defense against extracellular microbes, microbial toxins and foreign macromolecules.
• The cells involved in AMI are B-lymphocytes, TH cells and phagocytic cells.

Mechanism of Antibody mediated immune response:

• The effector response of antibody mediated immune response completes in three steps:
  • Activation and proliferation of B-cells to produce antibody
  • Class switching of antibody
  • Antibody mediated removal of antigen

Step I: Activation and proliferation of B-cells

• When the B-cell development and maturation completes in bone marrow, they are exported to the peripheral lymphoid tissues.
• When the mature B-cell in periphery is first exposed to antigen, they are activated by extensive cross-linkage of antigens to the mIg receptor on B-cells.
• The cross-linking of Ag with mIg generates signal 1 which leads to internalization of Ag-Ab complex by endocytosis and increase expression of class II MHC molecule with antigenic peptides and co-stimulatory receptor B7.
• Then the antigen presented with MHC-II on B-cell membrane is recognized by TCR of TH cell.
• This interaction plus co-stimulatory signal generated by B7-CD25 interaction activates the TH cells.
• The activated TH cell then begins to express CD40L.
• The interaction of CD40 and CD40L provides signal 2.
• The signal 2 along with co-stimulatory signal provided by B7-CD28 interaction stimulates the B-cell to begin expression of receptor for various cytokines.
• The binding of cytokines released by the TH cell to cytokines receptors in B-cell sends signals that supports the progression of B-cell to DNA synthesis and for the differentiation of B-cell into antibody producing plasma cell and memory cell.

Step II: Class switching of antibody

• In response to CD40 engagement and cytokines, some of the progeny of activated IgM and IgD expressing B-cell undergo the process of heavy chain isotype switching.
• It leads to the production of antibodies with heavy chain of different classes such as Ƴ, α and ε.
• Isotype switching occurs in peripheral lymphoid tissues.
• CD40 signaling and cytokinin produced by TH cell play essential role in regulatory switching to particular heavy chain isotypes.
• The major mechanism by which CD40 signals induce isotype switching is the induction of AID (Activation induced Deaminase) gene downstream of CD40.

Step III: Antibody mediated removal of antigen

• Antibody mediated removal of antigens consists of a number of effector mechanism.
• It needs the participation of various cellular and humoral component of immune system involving phagocytes and complements.
  • Neutralization
  • Antibody mediated opsonization and phagocytosis
  • Antibody dependent cell mediated cytotoxicity (ADCC)
  • Complement system

i. Neutralization of microbes and microbial toxins:

• The first step for removal of Ag is neutralization of microbes and toxins.
• Neutralization is the process by which antibodies against microbes and microbial toxins blocks the binding of these microbes and toxins to cellular receptor, thus inhibiting the infections.
• Neutralization of microbes by antibodies may occur by different ways.
• Microbes enter the host cells by binding of their particular surface molecule to host cellular receptor antibodies then bind to these microbial structures and interfere with the ability of microbes to interact with cellular receptors. This interaction is called steric hindrance.
• Very few antibodies bind to microbes and induce conformational change in the surface molecules that prevent the microbes from interacting with cellular receptor. This interaction is called allosteric effect of antibodies.
• Microbial toxins also cause pathogenic infection by binding to specific cellular receptors. Antitoxin (antibodies) sterically hinder the interaction of toxin to cellular receptor of host cell and prevent the tissue from injury and disease.

ii. Antibody dependent cell mediated cytotoxicity:

• NK cells and other leukocytes bind to antibody coated cells by the receptor and destroy these cells.
• This process is termed as antibody dependent cell mediated cytotoxicity.
• FcƳR III is present on the NK cells which binds to antibody coated cells.
• Since, FcƳR III is a low affinity receptor, it can bind only IgG bound infected cells but does not bind circulating monomeric IgG.
• Thus, ADCC takes place only when the target cell is coated with antibody molecules.
• Interaction of FcƳR of NK cell with IgG coated target cells, activates the NK cell to synthesize and secrete cytokines such as IFN-Ƴ and discharge the contents of their granules.
• This leads to lysis of antibody coated cells.
• Eosinophils, mediate a special type of ADCC directed against helminthic parasites.
• Helminths are too large to be engulfed by the phagocytes and their integument is relatively resistant to the microbicidal products of neutrophils and macrophages.
• They can be killed by a basic protein present in the granules of eosinophils.
• IgE coats the helminths and eosinophils can then bind to IgE through FcƳRI.
• The binding activates eosinophil to release their granules content which results in the killing of the helminths.

iii. Antibody mediated opsonization and phagocytosis:

• Mononuclear phagocytosis and neutrophils express a variety of surface receptors that directly bind microbes and ingest them and cause intracellular killing and degradation.
• Mononuclear phagocytosis and neutrophils express receptors for the Fc portion of IgG antibodies that specifically bind antibody coated (opsonized) particles.
• A product of complement activation called C3b can also opsonize microbes and are phagocytosed by binding to a leukocyte receptor for C3b.
• The process of coating particles for phagocytosis is called opsonization.
• Antibodies and complements proteins that perform their function are called opsonins.
• Phagocytosis of IgG coated particles is mediated by binding of the Fc portion of opsonizing antibodies to FcƳR on phagocytosis.
• Antibodies bound to antigens from multivalent arrays and are bound by phagocyte FcR with much higher avidity than free antibodies.
• Binding of multiple FcR to antibody coated particle lead to engulfment of the particles and their internalization in phagocytic vesicles.
• These phagosomes fused with lysosomes and the phagocytosed particles are destroyed in phagolysosome.
• Phagocytosis is also activated by the signal transducted by binding of opsonized particles to FcƳR I.
• This signal result in activation of tyrosine kinase in the phagocytes, which stimulates production of various microbicidal molecules.
• Phagocyte activation leads to the production of enzyme phagocyte oxidase, which catalyse the intracellular generation of reactive oxygen intermediates that are cytotoxic to phagocytosed microbes.
• Too large microbes that cannot be phagocytosed are killed by the activation of leukocytes that is capable of extracellular killing by secretion of hydrolytic enzymes and reactive oxygen intermediates.

iv. Activation of Complement system:

• Complement system is a system of lytic enzyme found in blood.
• These lytic enzymes are usually inactive, they are activated only under particular condition to generate products that mediate various effector functions of complement.
• Activation of complement involves the sequential proteolysis of protein to generate newly assembled enzymes complex with proteolytic activity.
• Zymogens are termed as the proteins that gain proteolytic enzymatic activity by the action of other proteases.
• The process of sequential zymogen activation is an enzymatic cascade.
• Proteolytic cascade allows tremendous amplification because each enzyme molecule activated at one step can generate multiple activated enzyme at next steps.
• The products of complement activation become covalently attached to microbial cells surface or to antibodies bound to microbes and to other antigens.

Antibody Mediated Immunity (AMI): Activation and mechanism of antibody mediated antigen removal

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• Cell mediated immune response (CMI) is the host defense that are mediated by Antigen specific T-cells and various non-specific cells of immune system.
• It protects against intracellular bacteria, virus and cancer and is responsible for graft rejection.
• Both the antigen specific and non-specific cells can contribute to CMI.
• Ag-specific cells include CD8+ cytotoxic T-lymphocytes (Tc or CTL) and cytokine secreting CD4+ TH cells that mediate delayed type hypersensitivity.
• The non-specific cells include NK cells and non-lymphoid cells types such as macrophages, neutrophils and eosinophils.
• The activities of both specific and non-specific cytotoxic components of immunity depends on effective local concentrations of various cytokines.

Types of Cell mediated immunity on the basis of Effector response:

• Cell mediated immune responses can be divided into two major categories according to the different effector population that are mobilized.
• i.e. 1) Ag-specific CMI   2) Non-specific CMI

i) Antigen specific Cell Mediated Immunity: Steps

• The Antigen (Ag) specific CMI response includes the cytotoxic T lymphocytes (CTLs) as effector population.

Step I: Activation of T-cells or generation of effective CTLs

• Nerve Tc cells or generation of killing target cells only after a naïve T-cell has been activated will differentiate into a functional CTL with cytotoxic activity.
• The activated of T-cell into CTL requires at least three sequential signals.
• An antigen specific signal transmitted by TCR complex upon recognition of a peptide-class-I MHC molecule complex on an APC.
• A co-stimulatory signal transmitted by the CD28 and B7 interaction of Tc cells and APC.
  – This signal stimulates the expression of IL-2R and lesser amount of IL-2 on Tc cell.
• A signal induced by the interaction of IL-2 with the high affinity IL-2R resulting in the proliferation and differentiation of antigen activated Tc cells into effector CTLs which consist of 65kDa monomer of protein called perforin and several serine proteases called granzymes.

Step II: Lysis of target cell in Antigen mediated cell immunity:

• The process of cell lysis begins when TCR-CD3 complex on CTL recognizes antigen associated with class-I MHC on the target cell.
• After this antigen-specific recognition, the integrin receptor LFA-I on the CTL binds to the ICAM on the target cell resulting in the formation of a conjugate.
• Ag mediated CTL activation converts LFA-I from low affinity state to high affinity state.

Stages in Antigen specific Cell mediated killing of target cells

i. Membrane attack:

• Immediately after the formation of CTL-target cell conjugate, golgi stacks and storage granules concentrate near the junction with the target cell.
• The perforin monomers and granzymes protease are then released from the granules by exocytosis into the space at junction between the two cells.
• Then the granzymes get entry into the target cell.
• There are two ways of entry of granzymes into the target cells.
  1. When the perforins contact the target membrane, they undergo a conformational change, exposing an amphipathic domain that inserts into the target cell membrane, then polymerize in the presence of Ca²⁺ to form cylindrical pores with an internal diameter of 5-20nm. After formation, granzymes enter through the pores into target cell.
  2. Many target cells have receptors known as mannose-6-phsophate on their surface that bind to granzyme B. Granzyme B-mannose-6-phosphate receptors complexes are internalized and appear inside the vesicles. Perforin internalizes at same time, then forms pore that release granzyme B from the vesicles into the cytoplasm of the target cell.

ii. Cytotoxic T-lymphocytes (CTL) dissociation:

• The LFA-I persists in the high affinity state for only 5-10 minutes after antigen mediated activation and then it returns to the low affinity state.
• This downshift in LFA-I affinity facilitates dissociation of CTL from the target cell.

iii. Target cell destruction:

• Once it enters the cytoplasm of the target cells, granzyme B initiates cascade of reaction that results in the fragmentation of target cell DNA into 200bp oligomer.
• This type of DNA fragmentation is typical of apoptosis.
• Granzymes which are protease don’t directly mediate DNA fragmentation, rather, they activate an apoptotic pathway within the target cell.
• Some, CTL lack perforin and granzymes B.
• In these cases, cytotoxicity is mediated by FAs.
• The FAs ligand, FasL is found on the membrane of CTL and interaction of FasL with FAs on target cell triggers apoptosis.

iv. Apoptosis:

• The feature of cell death by apoptosis is the involvement of the caspase family.
• Normally, caspases are present in the cell as inactive pro-enzyme, procaspase which requires proteolytic cleavage for conversion to active form.
• More than a dozen caspases have been found each with its own specificity.
• Cleavage of procaspase produces activator (active initiator caspase), which cleaves other procaspases, thereby activating their proteolytic activity.
• CTL uses granzyme and FasL to initiate caspase cascade in their target.
• Granzymes introduced into target cell mediate proteolytic event that activate an initiator caspase.
• Similarly, the engagement of Fas and FasL causes the activation of an initiator caspase in target cell.
• Fas is associated with FADD which in turn associates with procaspase, type of caspase 8.
• On Fas crosslinking, procaspase 8 is converted to caspase 8 and initiates an apoptotic caspase cascade.

ii) Non-specific Cell Mediated Immunity:

• Non-specific CMI response includes NK cells, macrophage, neutrophil and eosinophil.
• These cells can use antibody as receptor to recognize and for billing the target cells.
• NK cells appear to kill tumor cells and virus infected cells by processes similar to those employed by CTLs.
• NK cells possess FasL on their surface and subsequently induce death in Fas-bearing target cells.
• The cytoplasm of NK cell has many granules containing perforin and granzymes unlike CTL, which must be activated before granules appear.
•  NK cells are constitutively cytotoxic and always have granules.
• After an NK cell adheres to target cell, degranulation occurs with release of perforin and granzymes at the junctions of interacting cells.
• Perforin and granzymes play same role in NK-mediated killing of target cell by apoptosis as they do in CTL-mediated killing process.

Antibody dependent Cellular Cytotoxicity (ADCC):

• A number of cells that have cytotoxic potential express membrane receptors for the Fc region of the antibody molecule when antibody is specially bound to a target cell, cells bearing the Fc receptors can bind to a target cell, subsequently causing lysis of the target cell.
• Although, the cytotoxic cells are non-specific for antigen, the specificity of antibody directs them to the specific target cell. This type of cytotoxicity is known as ADCC.
• Target cell killing by ADCC appears to involve a no. of different cytotoxic mechanism but not complement mediated lysis.
• When macrophage, neutrophil, eosinophil bind to target cell by Fc receptors, they become more active metabolically, as a result, the level of lytic enzymes in their cytoplasmic lysosome or granule increases.
• Release of these lytic enzymes of side of Fe mediated contact results in the damage of target cell.
• In addition, activated monocytes NK cells and macrophages secrete TNP that have cytotoxic effect on the bound target cell.
• Since NK cells and eosinophils contain perforin in the cytoplasmic granules, their target killing also involves perforin mediated damage.

Cell mediated immunity (CMI): Antigen Specific and Non-specific CMI

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• The development of plasma cell and memory B cells can be divided into three broad stages:
• Generation of mature, immunocompetent B-cells (maturation)
• Activation of mature B-cells and the differentiation of the activated B-cells, into plasma cells and memory B cells.
• These three stages can be divided into two phases:
  1. Antigen independent phase:
     • This takes place in bone marrow.
     •  It involves the maturation of lymphoid progenitors to matured naive B cells.
  2. Antigen dependent phase:
     • This takes place in lymph node.
     • It involves activation of mature B-cells then they encounter antigen and their differentiation into plasma cells and memory B-cells.

B-cell maturation:

• The generation of B-cell first occurs in embryo and continues throughout life.
• Before birth, the yolk sac, foetal liver and foetal bone marrow are the major sites of B cell maturation.
• After birth, the generation of mature B-cells occur in the bone marrow from hematopoietic stem cells (HSC).
• The HSC first divide to form lymphoid progenitor cells which then differentiate into the progenitor B-cells (pro B) which express a transmembrane tyrosine phosphatase called CD45R and signal transducing molecule Igα/ Igβ which are found associated with the membrane bound antibody in later stages of development.
• Pro-B cell also express CD19 (part of co-receptor), CD43 (leukosialin), CD24 (heat stable), and C-kit are present on the surface of Pro-B-cell.
• The pro-B-cells proliferate within bone marrow filling extravascular spaces between large sinusoids in the shaft of a bone proliferation of pro-B-cells to precursor-B-cells (pre-B-cell) require micro-environment provided by the bone marrow stromal cells.
• The stromal cell plays two important roles, they interact directly with Pro-B cell and Pre-B cell and they secrete various cytokines, notably IL-7 that support developmental process.
• Pro-B-cells need direct contact with stromal cells in the bone marrow during the earliest developmental stage.
• This interaction is mediated by several cell adhesion molecules including VLA-4 on Pro-B cell and its ligand, VCAM-1, on the stromal cell.
• After initial contact is made, a receptor on Pro-B cell called C-kit interacts with a stromal cell surface molecule known as stem cell factor (SCF).
• This interaction activates C-kit, a tyrosine kinase and Pro-B cell begins expressing receptor for IL-7.
• The Pre-B-cell express many of same marker that were present on Pro-B-cell, however they cease to express C-kit and CD43 and begin to express CD25.
• The IL-7 secreted by stromal cells drives the maturation process eventually inducing down the regulation of adhesion molecule on Pre-B cell.
• So, the proliferating cell can detach from stromal cells.
• At this stage, Pre-B-cell no longer requires direct contact with stromal cell but continues to requires IL-7 for growth and maturation.

Ig-gene re-arrangement producing immature B-cells:

• B-cell maturation depends on rearrangement of immunoglobulin DNA in the lymphoid stem cells.
• The first Ig-gene re-arrangement to occur in Pro-B-cell stage is a heavy chain DH-JH gene re-arrangement, this is VH-DH-JH rearrangement.
• If the first heavy chain rearrangement is not productive, then VH-DH-JH rearrangement continues on the other chromosome.
• Upon completion of heavy chain arrangement, the cell is classified as Pre-B-cell.
• Continued development of a Pre-B-cell into an immature B-cell requires a productive light-chain gene re-arrangement.
• Only one light chain isotype is expressed on the membrane of a B-cell because of allelic exclusion.
• Upon completion of productive light chain re-arrangement, it commits the immature B-cell to a particular antigenic specificity.
• This specificity is determined by the cells heavy chain VDJ sequence and light chain VJ sequence.
• Immature B cell expresses mIgM on its cell surface.
• The bone marrow phase of B-cell development culminates in the production of IgM bearing immature B-cell.
• At this stage of development, B-cell is still not fully functional.
• Thus, antigen induces death or unresponsiveness rather than division and differentiation.
• The co-expression of IgD and IgM on the membrane signals the full maturation.
• This progression involves a change in RNA processing of the heavy chain primary transcript to permit the production of two mRNAs, one encoding the membrane form of the µ chain and other encoding the membrane of the 𝛿 chain.

B-cell proliferation and activation:

• After export of B-cell from the bone-marrow, activation, proliferation and differentiation occur in the periphery and require antigen.
• Depending on the nature of the antigen, B cell activation proceeds by two different routes, one dependent of TH cell, the other not.
• The B cell response to thymus dependent (TD) antigen requires direct contact with TH cell, not simply exposure to TH derived cytokines.
• Antigens that can activate B cells in absence of this kind of direct participation by TH cells are known as thymus independent (TH) antigen.
• The TI antigens are divided into two types 1 and 2 and they activate B-cells by different mechanisms.
• Most TI1 antigens are polyclonal B cell activator i.e. they are able to activate B-cell regardless of their antigenic specificity.
• At high concentration TI-1 antigens will stimulate proliferation and antibody secretion by as many as one third of B-cells.
• It includes bacterial cell wall components including lipopolysaccharide.
• B cells are activated by TI-2 antigens by extensively crosslinking the mIg receptor.
• However, TI-2 antigens contrasts to TI-1 antigens in three important respects.
• First, they are not B-cell mitogens and do not act as a polyclonal activators.
• Second, TI-1 antigens activate both mature B-cells and immature B cells. Whereas TI-2 antigen activates mature B cells and inactivates immature B-cells.
• Third, although B cell response to TI-2 antigen does not require direct involvement of TH cells, cytokines derived from TH cells are required for efficient B-cell proliferation and for class switching to isotypes other than IgM.
• It includes highly repetitious molecules like bacterial flagellin.
• Activation of B-cell by soluble protein antigen requires the involvement of TH cells.
• Binding of antigen to B-cell mIg does not itself induce on effective competence without additional interaction with membrane molecule on the TH cell.
• In addition to it, a cytokine mediated progression is required for B-cell proliferation.

i. Formation of T-B conjugate:

• After binding of antigen by mIg on B cell, the antigen is internalized by receptors mediated endocytosis and processed within the endocyte pathway into peptide.
• Antigen binding also initiates signaling through the BCR that induces the B-cells to upregulate a no. of cell membrane molecules, including class II MHC molecules and co-stimulatory ligand B7.
• Increased expression of both of these membrane proteins enhance the ability of B-cell to function as an antigen presenting cell (APC) in TH cell activation.
• Once the TH cell recognizes a processed antigenic peptide displayed by a class II MHC molecule on the membrane of B-cells, the two cells interact to form a T-B conjugate.
• This structural adjustment facilitates the release of cytokines towards the antigen specific B-cells.

ii. CD40-CD40L interaction:

• Formation of a T-B conjugate not only leads to directional release of TH cell cytokines but also to the upregulation of CD40L, a TH cell membrane protein that then interacts with CD40 on the B-cell to provide essential signal for T-cell dependent B-cell activation.
• Interaction of CD40L with CD40 on the B-cell delivers a signal (signal-2) to the B-cell that in cooperation with the signal generated by mIg cross-linkage (signal1), drives the B cell into G1.
• The signal from CD40 are transducted by a no. of intracellular signaling pathway, ultimately resulting in changes in gene expression.

iii. Signals provided by TH cell cytokines:

• The antigenic specific interaction between a TH and a B cell induces a redistribution of TH cell membrane proteins and cytoskeletal elements that results in the polarized release of cytokines towards the interacting B-cell.
• Once the B cell becomes activated, it begins to express membrane receptors for various cytokines such as IL-2, IL-4, IL-5 and others.
• These receptors then bind the cytokines produced by these cytokine-receptor interaction support B-cell proliferation and can induce differentiation, proliferation and can induce differentiation into plasma cells and memory cells, class switching and affinity maturation.
  • Antigen crosslinking mIg, generating signal 1 which results in increased expression of class II MHC and co-stimulatory B7. Antigen-antibody complexes are internalized by receptor mediated endocytosis. Then it is degraded to peptides, some of which are bound by class II MHC and presented on the membrane as peptide-MHC complexes.
  • TH cell recognizes antigen-MHC-II on B-cell membrane. This plus co-stimulatory signal activates TH cell.
  • i. TH cell begins to express CD40L
    ii. Interactions of CD40 and CD40L provides signal 2
  • iii. B7-CD28 interactions provide co-stimulation to the TH cell.
  • i) B-cell begins to express receptors for various cytokines.
    ii) Binding to cytokines released from TH cell in a directed manner relays signal that the progression of the B-cell to DNA synthesis and to differentiation.

B-cell development: Maturation, activation and differentiation

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• The migration of progenitor T-cells from the early sites of hematopoiesis to the thymus takes place at about day 11 of gestation in mice and in 8^th or 9^th week of gestation in humans.
• T-cell maturation involves the re-arrangement of the germ-line TCR genes and the expression of various membrane markers.
• In thymus, the developing T cells are termed as thymocytes.
• These thymocytes proliferates and differentiates along developmental pathways that produce functionally distinct sub-population of mature T-cells.
• T-cells development initiates with the arrival of small numbers of lymphoid precursors migrating from the blood into the thymus where they proliferate, differentiate and undergo selection process that result in the development of mature T cells.
• When T-cells precursor arrive at thymus, they don’t express the signature surface markers of T cell as the T-cell receptors, the CD₃ complex or the co-receptors CD₄ and CD₈.
• In-fact these progenitor cells have not yet re-arranged their TCR genes and do not express the proteins, such as RAG-1 and RAG-2 that are required for re-arrangement.
• After arriving in the thymus T cell precursors enter the cortex and slowly proliferate.
• The differentiating T-cell pass through a series of stages that are marked by characteristic changes in their cell surface phenotype, during approximately three weeks of development in thymus.
• The thymocytes early in the development lack detectable CD₄ AND CD₈.
• As these cells are CD₄-, CD₈-, they are termed as double negative (DN) cells.
• In-fact DN-T cells can be sub-divided into 4 subsets (DN1-4) characterized by the presence or absence of cell surface molecules in addition to CD₄ and CD₈, such as:
  •  C-kit, the receptor for stem cell growth factors
  • CD₄₄, an adhesion molecule
  • CD₂₅, it is the alpha chain of the IL-2 receptor
• The cells that enter the thymus DN1 cells are capable of giving rise to all the subsets of T cells and are phenotypically C-kit⁺, CD₄₄ high and CD₂₅-.
• Once the DN₁ cells encounter the thymic environment, they begin to proliferate and express CD₂₅ becoming C-kit⁺, CD₄₄ high and CD₂₅+.
• These cells are called DN₂ cells.
• During the critical DN₂ stage of development, rearrangement of the genes for the TCR, Ƴ, 𝛿 and β chain begins.
• However, the TCR α locus does not re-arrange, presumably because the region of DNA encoding TCR α gene is densely compacted and not accessible to recombinase machinery.
• As cells progress to DN₃, the expression of both C-kit and CD₄₄ is turned off and TCR Ƴ, TCR 𝛿 , and TCR β re-arrangement progresses.
• Cells destined to become Ƴ𝛿  T cell, diverge at the transition between DN₂ and DN₃ and become mature.
• Ƴ𝛿  T cell which vary few changes in their surface phenotype most DN₂ cells are destined to give rise to T cells.
• On assuming the DN₃ phenotype (C-kit^–, CD₄₄– and CD₂₅+), the cells halt proliferation and protein products of TCR β rearrangements are detected in the cytoplasm of these cells.
• The newly synthesized β-chain combine with 33-kDa glycoprotein known as the pre-T cell receptor or pre-TCR.
• Formation of pre-TCR activates a signal transduction pathway that has the following consequences:
• It indicates that a cell has made a productive TCR β-chain re-arrangement and signals its further proliferation and maturation.
• It suppresses further re-arrangement of TCR β-chain gives resulting in the allelic exclusion.
• It renders the cell permissive for re-arrangement of the TCR α chain.
• It induces developmental progression to CD₄₊ and CD₈₊ double positive (DP) state.
• After β-chain rearrangement is completed the DN₃ cells quickly progress to DN₄, the level of CD₂₅ falls and both CD₄ and CD₈ co-receptors are expressed.
• Thus, double positive (DP) stage is a period of rapid proliferation.
• However, TCR α-chain gene rearrangement still has not occurred at this stage of time.
• The rearrangement of α-chain gene does not begin until the double positive (DP) thymocytes stops proliferating and RAG-2 protein level increases.
• T-cell development is a costly process for the host.
• An estimated 98% of all thymocytes do not mature i.e. they die by apoptosis within the thymus either because they fail to make a productive TCR gene re-arrangement or because they fail to survive thymic selection.
• Double positive thymocytes that express αβ TCR CD₃ complex and survive thymic selection develop into immature single positive CD₄+ thymocyte or single positive CD₈+ thymocytes.
• These single positive cells undergo additional negative selection and migrate from the cortex to medulla, where they pass from the thymus into the circulatory system.
• After the gene rearrangement, T-cell (Thymocytes) undergoes thymic selection.

Thymic selection of T-cell:

• The most characteristic property of mature T-cells is that they identify only foreign antigen combined with self MHC molecules.
• For this purpose, the thymocyte undergo two selection process in thymus.
• Positive selection:
  • Positive selection occurs in the cortical region of the thymus.
  • It involves the interaction of immature thymocytes with cortical epithelial cells.
  • This interaction allows the immature thymocytes to receive a protective signal.
  •  This signal prevents them from undergoing cell death.
  •  Cells whose receptors are not able to bind MHC molecules would not encounter with the thymic epithelial cells and as a result it would not receive the protective signal resulting their death by apoptosis.
  • This results in MHC restriction.
• Negative selection:
  • The population of MHC restricted thymocyte that survive positive selection includes cells with receptors having a range of affinities from low to high for self-antigen presented by self-MHC molecule.
  • Thymocytes with high affinity receptors are weeded out during negative selection via an interaction with thymic stomal cells.
  • In case of negative selection, dendritic cells and macrophages having class I and class II MHC molecules interact with thymocyte bearing high affinity receptors for self MHC molecule alone.
  • cells that undergo negative selection are observed to undergo death by apoptosis.
  • Tolerance to self-antigens encountered in the thymus achieved by eliminating T-cells that are reactive to these antigens.

T-cell activation:

• The central event in the generation of both humoral and cell-mediated immune responses in the activation and clonal expansion of T-cells.
• T-cells activation is initiated by interaction of the TCR-CD₃ complex with a processed antigenic peptide bound to either class-I (CD₈+ cell) or class II (CD₄+ cell) MHC molecules on the surface of an antigen presenting cells.
• The interaction and the resulting activating signals also involve various accessory membrane molecules on the T-cell and the antigen presenting cell.
• Interaction of T cell with antigen initiates cascade of biochemical event that induces the resting T-cell to enter the cell cycle, proliferating and differentiating into memory cells or effector cells.
• The key element in the initiation of T cell activation is the recognition by the TCR of MHC peptide complexes on antigen presenting cells.
• This event catalyses a series of intracellular events beginning at the inner surface of the plasma membrane and culminating in the nucleus, resulting in the transcription of genes that drive the cell cycle and/or differentiation of T-cell.

T-cell differentiation:

• CD₄+ and CD₈+ T-cells leave the thymus and enters the circulation as resting cells in the G₀ stage of cell cycle.
• There are about twice as many CD₄+ T-cells as CD₈+ T-cells in the periphery.
• T cells that have not yet encountered antigen (naive T-cells) are characterized by condensed chromatin, very little cytoplasm, and little transcriptional activity.
• Naive T cells continually reticulate between the blood and the lymph system.
• During recirculation, naïve T cells reside in secondary lymphoid organs/tissues such as lymph nodes.
• If a naive cell does not encounter antigen in a lymph node, it exits through the efferent lymphatics ultimately draining into the thoracic duct and rejoining the blood.
• It is estimated that each naive T cell recirculates from blood to the lymph nodes and back again every 12-24 hrs.
• This large-scale recirculation increases the chances that naive T cell will encounter appropriate antigen because only about 1 in 10⁵ naive T -cell is specific for any given antigen.
• If naive T cell recognize an antigen-MHC complex on an appropriate antigen cell or target cell, it will be activated, initiating a primary response.
• About 48hrs after activation the naïve T cells enlarges into a blast cell and begins undergoing repeated rounds of cell-division.
• Activation depends on a signal induced by the enlargement of the TCR complex and a stimulatory signal induced by the CD₂₈-B₇ interaction.
• These signals trigger entry of T cell into the G₁ phase of the cell cycle and at the same time, induce transcription of the gene for IL-2 and α chain of the high affinity IL-2 receptor (CD₂₅).
• Moreover, the co-stimulatory signal increases the half-life of the IL-2 mRNA.
• The increase in IL-2 transcription together with the stabilization of the IL-2 mRNA, increases IL-2 production by 100 folds in the activated T-cell.
• Secretion of IL-2 and its subsequent binding to the high affinity IL-2 receptor induces the activated naïve T cell to proliferate and differentiate.
• T-cells activated in this way divide 2-3 times per day for 4-5 days generating a clone of progeny cells which differentiates into memory or effectors T-cell populations.

T-cell maturation, activation and differentiation

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• The process of transfer of cells, tissues, or organs from one location to another with a motive of either repairing or replacing damaged or diseased organs and tissues is defined as transplantation.
• In case of failure of organ system or damage, it can be replaced by the healthy organ or tissue donated by donor.
• Transplantation is life savior procedure and is employed only when other treatment options fail.
• However, the immune system is the one to play a significant role in transplantation.
• The successful transplantation is all dependent on the complex mechanisms of immunity.

Types of grafts:

The following terms indicate different types of transplants:

1. Autograft:

• In this type of graft, the tissue is transferred from one body site to another in the same individual.
• For example, use of healthy blood vessels to replace blocked coronary arteries.

2. Isograft:

• The transfer of tissue between genetically identical individuals is termed as isograft.
• For Example the transfer of kidney from one identical twin to the other.

3. Allograft:

• In this type of graft, the tissue transfer takes place between genetically non-identical members of same species.
• Example: skin transplant between two individuals of different genotype.

4. Xenograft:

• The transfer of tissue or organ between different species is termed as xenograft.
• For example: the graft of monkey’s heart into a human.

5. ABO incompatible transplant:

• ABO is common term for blood group, which differs among individuals.
• The key strategy applied for the minimization of transplant rejection is the matching of blood group between donor and recipient.
• However, the compatibility is always not required for transplantations. For instance, ABO transplants can be carried out in children with immature immune systems with minimized risk of transplant rejection.

6. Stem cell transplant:

• Stem cells are capable of giving rise to indefinitely more cells of same type, and from which different other cells arise by differentiation.
• Hematopoietic stem cell transplants are done to replace damaged or worn out blood cells. Also, it is employed to treat various forms of cancer, eg. Leukemia.
• These stem cells can be obtained either from cord blood or from bone marrow.

Transplant rejection:

• Immune system works to recognize the foreign microbes or foreign threats and destroys them creating a barrier for the transplantation.
• When the immune system identifies the transplant as a foreign, it initiates a response that ultimately degrades the transplanted organ or tissue.
• This rejection caused by immune system in case of transplantation is termed as transplant rejection.
• The organ or tissue to be transplanted is termed as graft.
• Depending on the type of graft being transplanted and the genetic variance between the donor and the recipient, the intensity of the immune response varies accordingly.
• Thus, to prevent graft rejection, both the donor and the recipient are carefully matched for immune compatibility before the transplantation.
• The immune system can be manipulated for long term survival of the graft which ensures successful transplantation.

Immunology of transplant rejection:

• When the immune system confronts a foreign organism, it prepares for attack against it in order to protect the body from infection.
• It is must that the immune system should be able to distinguish between own healthy cells/tissues and foreign substances.
• Foreign invaders appear in form of small molecules termed as antigens.
• These molecules, when presented to the immune system, triggers the immune response.
•  It stimulates the production of antibodies specific to those antigens and amplifies the immune response.
• The group of genes that encodes proteins which identifies foreign agents to the immune system is termed as the Human Leukocyte Antigen (HLA) complex.
• These proteins act as ‘self-markers’ as they convey immune system not to trigger a response and are present on the surface of every cells.
• On the basis of genetic makeup, each individual shall have their own specific set of HLA proteins.
• Any cell unable to display HLA proteins will be identified as non-self by the immune system and will be further responded.

Mechanism of rejection:

• The term histocompatibility is used to represent the degree of similarity between the HLA genes of the donor and the recipient.
• The compatibility between the donor and the recipient depends on the similarity of genetic makeup between them.
• However, there will always be some extent of rejection even if the donor and recipient are genetically identical.
• Non-self HLA proteins, other surface proteins on the donor graft can also be recognized as a foreign antigen and forbid an immune response.
• In some cases, a patient encounter ‘graft versus host reaction’ where mature immune cells already available in the donor graft attacks the healthy cells of the recipient.

Clinical stages of transplant rejection:

1) Hyperacute rejection:

• The presence of pre-existing antibodies of the recipient, that match the foreign antigens of the donor, triggers an immune response against the transplant and results in this type of rejection.
• These antibodies could have been produced due to result of previous blood tranfusions, previous transplantations or multiple pregnancies.
• This takes place within minutes or hours after a transplantation.
• The blood clotting takes place, when antibodies react with cells in the blood vessels of the graft, which will prevent blood supply from the graft yielding an immediate rejection of the transplant.

2. Acute rejection:

• This takes place within the first 6 months after transplantation.
• Exception to identical twins, there prevails some degree of acute rejections in all transplantations.
• There is a high risk for the first 3 months for recipients, however rejection can still take place at a later stage.
• After the detection of non-self antigens, the formation of antibodies causes acute rejection.
• By suppressing the immune system, acute rejection can be treated to some extent and the permanent damage to the graft can be avoided in some cases.

3. Chronic rejection:

• Recurrence of acute rejection can definitely lead to chronic rejection of the graft resulting the failure of transplant.
• The exhibition of chronic rejection takes place as scarring of the tissue or organ which can occur for months to years after acute rejection has subsided.
• There is no cure for chronic rejection except the removal of graft till date.

Transplant immunology: Types of graft, and transplant rejection

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• Immunotherapy is defined as one of the methods of treatment by inducing, enhancing, or suppressing an immune response.
• It is one of the types of biological therapy in which the substances obtained from living organisms are used to treat cancer.
• Generally, immunotherapies are classified into two groups:
  • Activation immunotherapies: includes immunotherapies employed for amplifying an immune response.
  • Suppression immunotherapies: includes immunotherapies that suppress or reduce an immune response.
• Mostly, immunotherapies have been found to be promising to cure varieties of cancer.
• Also, it often has fewer side effects in comparison to the existing drugs.

Relation between immunotherapy and immune system:

• Immunotherapy helps to enhance immune cells by providing with additional components.
• Immunotherapy instructs the immune system to identify and defend specific cancer cells.
• Immunotherapy helps to eradicate cancer by boosting immune cells.

Types of Immunotherapy for cancer:

There are various types of immunotherapy. They are listed as follows:

1. Monoclonal antibodies and tumor-agnostic treatments, such as checkpoint inhibitors
2. Oncolytic virus therapy
3.  CAR T-cell therapy
4. Cancer vaccines
5. Immune checkpoint inhibitors

1. Monoclonal antibodies (mAbs or MoAbs) and tumor-agnostic treatments:

• When a threat is sensed by the immune system, it produces antibodies.
• Antibodies are proteins that interact with antigens and initiate an immune response in the body.
• Monoclonal antibodies are defined as antibodies that are prepared in laboratory in order to boost the natural antibodies or to defend the foreign threats itself.
• Monoclonal antibodies help to fight cancer in several ways, for instance, monoclonal antibodies can be designed to fight with a specific cancer cell.
• Thus, it is also termed as targeted therapy.
• Basically, three types of mAbs are introduced, they are:
  • i. Naked monoclonal antibodies:
    • Most commonly used for treatment of cancer.
    • They are not attached to any other substance.
    • These antibodies instruct the immune system to defend cancer cells or block proteins within tumor.
  • ii. Conjugated monoclonal antibodies:
    • These are attached with either a radioactive particle or chemotherapy drug.
    • They are capable of attaching directly to the cancer cells.
    • It works by delivering the drug to the location of utmost requirement.
    • There are reduced or less chance of side effects and enhances the chemotherapy as well as radiation.
  • iii. Bispecific monoclonal antibodies (BsMAb):
    • These are antibodies designed in order to bind with two proteins at once.
    • Few can bind to both cancer cells and an immune system cell facilitating the immune response on cancer.

2. Oncolytic virus therapy:

• In this type of treatment, viruses used are modified in the lab with a motive to infect and kill specific tumor cells.
• Procedure of Oncolytic virus therapy:
  • First, the genetically modified virus is injected into the tumor.
  • Then the virus reaches the cancer cells and make copy of itself.
  • This leads to the disruption of cancer cells and ultimately their death.
  • As soon as the cells die, it stimulates the immune system to attack any cancer cells on the body having alike proteins as that of dead cells.
• This modified oncolytic virus doesn’t affect healthy cells.
• This therapy is considered superior to all other immunotherapies as it does not depend on any specific antigen expression patterns.
• The features that makes it an ideal candidate for the treatment of diverse malignancies are as follows:
  • Oncolytic viruses enhance the recruitment of tumor-infiltrating lymphocytes (TILs)
  • Reprogramming of immunosuppressive tumor micro-environment (TME)
  • Boosts systemic anti-tumor immunity

 3. CAR T-cell therapy:

• It stands for chimeric antigen receptor T-cell therapy (CAR T-cell therapy).
• T cell, a type of leukocytes is one of the main constituents of the adaptive immune system.
• In CAR T-cell therapy, the doctor reprograms T cells after taking out T cells from blood in order to find cancer cells more easily in contrast to it, T-cell therapy instruct the T-cells to search for tiny bits of specific antigens inside the cancer cells.
• Procedure of CAR T-cell therapy:
  • At first T cells are isolated from patient’s blood.
  • Then, the receptors, which are specific proteins are added to it in the laboratory.
  • The receptors permit the T cells to identify cancer cells.
  • Then, the programmed T cells are then re-entered into the body.
  • Now, these cells find and destroy cancer cells.
• Side effects such as low blood pressure, fever, confusion, and in rare cases, seizures are observed.

4. Cancer vaccines:

• These are also termed as therapeutic vaccines.
• The vaccines are employed to people already diagnosed with cancer with a motive to increase body’s natural defense in order to fight cancer.
• The vaccines may either prevent the cancer from recurrence, destroy any cancer cells remnant even after the accomplishment of other treatments or stop a tumor from spreading.
• Procedure of cancer vaccine therapy:
  • As we know, when the antibodies are produced in response to the antigens, the immune system develops memory cells, which will respond to these antigens in future.
  • Cancer vaccines enhance the immune system’s capacity to identify and destroy antigens.
  • Certain molecules termed as cancer specific antigens are present on the surface of the cancer cells, which are lacked by healthy cells.
  • These molecules act as antigens when given to a person, and trigger the immune system to identify and kill cancer cells having these molecules on the surface.
  • Few cancer vaccines also consist of adjuvants that may enhance the immune response.

5. Immune-checkpoint inhibitors:

• It is the significant function of immune cells to be able to differentiate between the own normal cells and foreign cells.
• Checkpoints are thus required, in order to monitor the foreign cells.
• In general term, immune checkpoints are molecules on specific immune cells that needs to be either activated or inactivated for starting an immune response.
• Drugs targeting these checkpoints are found to be promising for cancer treatment.
• These drugs are hence termed as checkpoint inhibitors.
• Two types of checkpoint inhibitors are described on the basis of proteins they target, they are:
• i. Checkpoint inhibitors that target PD-1 or PD-L1:
  • T cells are immune cells and PD-1 is a checkpoint protein on it.
  • PD-1 protein prevents the T-cells from attacking other cells in the body, acting as a type of off switch.
  • It occurs when it is bound to PD-L1, which is a protein on some normal and cancer cells.
  • After the binding, it stops T-cells from attacking any cells favoring the cancer cells with high PD-L1 protecting it from immune attack.
  • Monoclonal antibodies targeting either PD-1 cells or PD-L1 cells can prevent the binding and enhance immune response against cancer cells.
    • Drugs targeting PD-1 (PD-1 inhibitors):
      • Pembrolizumab (Keytruda)
      • Nivolumab (Opdivo)
      • Cemiplimab (Libtayo)
    • Drugs targeting PD-L1 (PD-L1 inhibitors):
      • Atezolizumab (Tecentriq)
      • Avelumab (Bavencio)
      • Durvalumab (Imfinzi)
• ii. Checkpoint inhibitors that target CTLA-4
  • Some T cells contain CTLA-4 protein that also acts as a off switch inorder to regulate immune system.
  • The CTLA-4 is inhibited by Ipilimumab (Yervoy) which is a monoclonal antibody that attaches to CTLA-4.
  • The body’s immune response is hence improved.
  • This drug is proved to treat melanoma of skin.
• Side effects of checkpoint inhibitors include:
  • – Inflammation in the lungs
  • – Rashes along with itchiness
  • – Kidney infections
  • – Diarrhea

Immunotherapy-Types of Immunotherapy for cancer

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• Haematopoiesis is defined as the process of formation, development and differentiation of blood cells.
• The blood cells are formed from haematopoietic stem cells (HSCs) which are either multipotent or pluripotent in nature.
• In the prenatal stage, haematopoiesis occurs in the yolk sac during the first weeks of embryonic development and transitions to the spleen, liver, lymph nodes and finally in the bone marrow continuing for lifetime.

Hematopoietic stem cells (HSCs) and types

• Haematopoietic stem cells (HSCs) are special type of cell present in rar the bone marrow, they are rare and their numbers are strictly controlled by a balance of cell division, death, and differentiation.
• HSCs divide generating daughter cells. Some daughter cells retain the stem-cell characteristics of the mother cell having property of self self-renewing and able to give rise to all blood cell types. While other daughter cells differentiate into progenitor cell that lose their self-renewal capacity and giving rise to a particular blood cell lineage.
• Therefore, early in hematopoiesis, a multipotent stem cells differentiates along one of two pathways, giving rise to either a common lymphoid progenitor cells or a common myeloid progenitor cells
• Both the myeloid and lymphoid lineages are engaged in dendritic cell formation.
• Myeloid cells: It consists of-
  • Monocytes
  • Eosinophils
  • Basophils
  • Neutrophils
  • Macrophages
  • Erythrocytes
  • Megakaryocytes
  • Platelets.
• Lymphoid cells: It consists of-
  • B cells
  • T cells
  • Natural killer cells
  • Innate lymphoid cells.

The common myeloid progenitor cells:

• The common myeloid progenitor cell (CFU-GEMM) can differentiate into erythrocytes, thrombocytes, granulocytes, lymphocytes and monocytes.
• For the differentiation and proliferation of CFU-GEMM from HPCs, Interleukin-3 and Granulocyte-macrophage colony-stimulating factor (GM-CSF) are implicated.
• Stem cell factor (SCF) is a cytokine that can trigger the growth and total number of CFU-GEMM.
• CFU-GEMM is the precursor to several colony forming units which finally give rise to different blast cells.
• These consists of the following:
  • CFU-Meg (from which promegakaryoblasts are produced)
  • CFU-E (from which proerythroblasts are produced)
  • CFU-GM (both monoblasts and myeloblasts are derived from this type of colony forming unit)

Process of hematopoiesis:

• According to the monophyletic theory of hematopoiesis, the pluripotent stem cells multiply to produce more of the pluripotent stem cells, making sure of the steady and lasting supply of stem cells.
• Some of the pluripotent stem cells now differentiate into precursor cells that are least partially dedicated to form one type of mature blood cell.
• Pluripotent cells multiply at low pace into one of the five possible unipotential stem cells.
• These unipotential stem cells then multiply rapidly into the precursor of the destined specific mature blood cell.
• The typical process of hematopoiesis consists of the differentiation of the multipotential hematopoietic stem cell into either the common myeloid or lymphoid progenitor.
• Then, relying upon the cytokines and resulting transcription factors that are activated, the myeloid progenitor can differentiate into a myeloblast.
• This myeloblast leads to granulocyte (basophils, eosinophils, or neutrophils) or monocyte (macrophages and dendritic cell) development.
• Also, it leads to the differentiation of megakaryocytes into platelets, or erythroblasts into erythrocytes.
• Lymphoid dendritic cells can form directly from the common lymphoid progenitor.
• Furthermore, the differentiation of the common lymphoid progenitor into a lymphoblast results the further development of natural killer cells or lymphocytes (T and B cells).
• Once B cells get activated in secondary lymphoid organs, it further differentiate into plasma cells.
• These plasma cells secrete antibodies. 

Regulation of Haematopoiesis:

• Hematopoiesis is largely regulated by the presence of cytokines.
• These cytokines are responsible for regulating the differentiation of multipotential hematopoietic stem cells into specific cell types by the activation of transcription factors.
• The cytokines is very important for differentiation of particular cell types otherwise animal dies during embryogenesis.
• Some cytokines that regulates haematopoiesis are:
  • Granulocyte macrophage-colony stimulating factor (GM-CSF):
    • It enhances the myeloid lineage, finally leading to the differentiation of granulocytes and macrophages. Such cytokines are termed as growth factors.
    • These growth factors are needed throughout the process of hematopoiesis functioning in order to activate transcription factors.
  • Transcription factor GATA-2:
    • It is required for the development of all hematopoietic lineages; in its absence animals die during embryogenesis.
  • Transcriptional regulator Bmi-1:
    • It is required for the self-renewal of HSCs, and in its absence animals die within 2 months of birth because of the failure to repopulate their red and white blood.
• Other Examples of cytokines involved in haematopoiesis are:
  • Colony-stimulating factors (CSFs)
  • Erythropoietin (EPO)
  • Thrombopoietin (TPO)
  • Granulocyte colony-stimulating factor (G-CSF)
  • Monocyte colony-stimulating factor (M-CSF)
  • Tumor necrosis factor (TNF)
  • Transforming growth factor (TGF)
  • Stem cell factor (SCF)
  • Leukemia inhibitory factor (LIF)

Erythropoiesis:

• The process of formation of red blood cells termed as erythrocytes is known as erythropoiesis.
• It is enhanced by decreased levels of oxygen in the blood, which signals for the secretion of erythropoietin.
• Erythropoietin is a hormone central to the formation of red blood cells.
• Erythropoiesis takes on average 2 days to be completed from to form mature red blood cell from unipotential hematopoietic cell.
• 2 million erythrocytes are produced every second in our bodies.
• Hematopoietic cells determined to become red blood cells usually get smaller and more condensed as they mature until there is finally loss of their nuclei.
• The unipotential cell becomes proerythroblast, which has uncondensed nucleus and has basophilic or blue cytoplasm. 
• Then the cell becomes a basophilic erythroblast, which is followed by a polychromatophilic erythroblast stage.
• In  polychromatophilic erythroblast stage, the nucleus becomes more condensed than the latter two stages and the cytoplasm is reduced.
•  In the succeeding orthochromatophilic erythroblast stage, the nucleus is much smaller than that of the prior stages having a pinker cytoplasm.
• Then comes the reticulocyte stage, where the red blood cell lacks nucleus, but still stains somewhat blue because of the remnants of polyribosomes within the cell.
• Ultimately, the erythrocyte is the mature red blood cell, with no nucleus and no polyribosome remnants and as a result stains pink.

Granulopoiesis

• The process of formation of granulocytes is termed as granulopoiesis.
• Granulocytes are white blood cells having multi-lobular nuclei and cytoplasmic granules.
• The unipotential hematopoietic cell which forms a myeloblast is large.
•  It has a cytoplasm that stains blue with a large nucleus.
• This cell gives rise into a promyelocyte that contains azurophilic granules. Then it becomes a myelocyte, which has a non-indented still rather large nucleus.
• This cell then gives rise to a metamyelocyte, which is alike in size to a mature granulocyte and the nucleus starts to become indented.
• After this stage is the band cell stage, where the nucleus resembles a horseshoe and has definitive indentation.
• Ultimately, there is the mature granulocytes having a lobed nucleus and cytoplasmic granules.
• The entire process occurs over a period of 2 weeks.

Monopoiesis:

• The process by which monocytes are formed is termed as monopoiesis.
• The monoblast is the committed progenitor cell, found only in the bone marrow. Also, monoblast has a basophilic cytoplasm without granules.
• These monoblasts give rise to promonocytes, which are smaller in size with nuclei that become slightly indented, before becoming monocytes.
•  Monocytes have kidney-shaped nuclei and can develop into dendritic cells or macrophages.

Lymphopoiesis:

• The formation of lymphocytes, starts from their first committed progenitor cells, lymphoblasts, this process is called Lymphopoiesis.
• These cells after maturation, are able to differentiate into either B, T or natural killer cells.  

Thrombopoiesis:

• Megakaryocytes, which are extremely large cells within the bone marrow forms the platelets, this process is termed as thrombopoiesis.
• When the plasma membranes of megakaryocytes are fragmented, the origin of individual platelets take place, thus generating platelets containing many granules.

Hematopoiesis: Types of Haematopoietic stem cells, Process and Regulation

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