T Cell Receptor (TCR) Studies

T Cell Receptor (TCR) Studies

• Early studies of the T cell receptor were difficult due to its membrane-bound nature.
• The molecule responsible for T cell antigen recognition is a heterodimer with a single variable region like the B cell receptor.
• Alpha Beta TCRs have a high degree of specificity.
• Gamma delta T cells recognize specific PAMPs, functioning more like innate immunity.
• The antigen binding site is located at the hyper variable region, which differs from the constant regions that anchors the molecule to the cell membrane for intracellular signalling.

Gamma Delta T Cells

• Recognize specific classes of antigens.
• Function in a manner consistent with innate immunity.
• Diversify the immunological function of T cells.
• Hosts benefit from an expanded functional role of T cells to overcome infection.
• They are fewer in number in circulation than Alpha Beta T cells.

T Cell Co-receptors

• Co-receptors like CD4 and CD8 are required for proper T cell receptor functioning and interaction with MHC molecules.
• Alpha/Beta TCRs use CD4 or CD8 co-receptors.
• CD4 is used by T helper cells, which recognize antigens presented by MHC class II molecules.
• CD8 is used by cytotoxic T cells, which recognize antigens presented by MHC class I molecules.
• Gamma/Delta TCRs do not require CD4 or CD8 co-receptors for function.
• Gamma/Delta T cells recognize non-peptide antigens through mechanisms not involving the classical MHC presentation pathway.
• Have a broader range of target antigens, like lipids and stress molecules, presented by non-classical MHC molecules or other receptors.
• They participate in rapid, innate-like immune responses.

TCR Complex Signaling

• Contains variable regions, constant regions, and the CD3 complex.
• Signaling is facilitated by the CD3 complex, including CD247, containing ITAMs molecules.

Differences Between Alpha/Beta and Gamma/Delta TCR

• Gamma/delta T cells recognize specific antigen classes and function like innate immunity.
• There are fewer gamma/delta T cells than alpha/beta T cells in circulation.
• Circulating gamma/delta T cell specificity may target common pathogens.
• Gamma/delta cells do not use CD4 or CD8 co-receptors, are generated mostly in fetal life, and are less polymorphic in TCR structure.

TCR Alpha Beta Structure

• Composed of two polypeptides: an Alpha chain (49 kDa) and a Beta chain (43 kDa).
• Each chain has two extracellular domains, a transmembrane portion, and a cytoplasmic tail.
• The outermost domain is the variable domain (V), the other is the constant domain (C).
• Internal disulfide bonds stabilize each domain.
• Both alpha and beta chain variable domains form the antigen binding site.
• T cell receptors cannot initiate signaling alone due to extremely short cytoplasmic tail of about three amino acids.
• TCRs are associated non-covalently with a complex of proteins: gamma, delta, epsilon, and zeta.
• CD3 consists of heterodimers formed by delta and epsilon proteins, and gamma and epsilon proteins.
• Zeta chains linked together by a disulfide bond form the remaining part of this protein complex.

B Cell Receptor (BCR)

• Identifiable and well-studied due to soluble properties like immunoglobulin molecules.
• Membrane IG is associated with the structure of the B cell receptor.
• The disulfide linked heterodimer IG alpha and iG beta forms the BCR complex.
• Functionally divided into two antigen binding variable regions and constant regions.
• The discovery of this disulfide linked heterodimer, IG alpha and iG beta solved the long standing dilemma regarding VCR stability and structural capability of signal transduction

BCR Structure

• Contains a light chain, heavy chain, two variable regions, and IG alpha/IG beta.
• IG alpha/IG beta anchors the immunoglobulin molecule through the lipid bilayer.
• IG alpha/IG beta facilitates signal transduction, partially via tyrosine-based activation motifs (ITAMs).
• It has two antigen binding sites and the BCR is stabilized by the Igalpha and Igbeta heterodimer that also facilitates signal transduction, basically the same as the CD3 molecule.

BCR vs TCR

• BCR has two antigen recognition regions (immunoglobulins on the B cell surface serve as BCRs)
• T cells express membrane bound TCRs.
• T cells produce TCRs with higher affinity for antigen bound to MHC molecules, rather than soluble antigen.
• Both BCR and TCR utilize accessory molecules for anchoring and signal transduction e.g., IG alpha/IG beta or CD3 complex

TCR vs BCR

• TCR contains a single antigen binding site and binds only to peptide antigens presented on MHCs.
• BCR is a surface immunoglobulin with two antigen binding sites, heavy and light chains, and transmembrane proteins (Igbeta and Igalpha) connected to ITAM chains.
• Membrane-bound immunoglobulin (Ig)
• BCR consists of two identical heavy chains and two identical light chains, forming a Y-shaped structure
• BCR is anchored to the B cell membrane by a transmembrane region.
• The tips of the Y-shaped structure contain variable regions that bind specifically to antigens.
• Transmembrane protein make up the T cell receptor
• TCR consists of an α and β chain (or γ and δ chains in some T cells), forming a heterodimeric or homodimeric structure.
• TCR is Embedded in the T cell membrane by a transmembrane region.
• The tips of the TCR chains contain variable regions responsible for antigen recognition.

BCR - Antigen Recognition

• BCR Recognizes antigens directly, including proteins, carbohydrates, and lipids.
• Expressed on the surface of B cells.
• BCR initiates the activation of B cells when antigens bind to its variable regions.
• Activated B cells differentiate into plasma cells, which produce antibodies that neutralize or eliminate pathogens.

TCR - Antigen Recognition

• TCR recognizes antigens presented by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs).
• Expressed on the surface of T cells.
• TCR plays a central role in T cell activation when it binds to antigen-MHC complexes.
• Activated T cells differentiate in effector T cells with various functions, such as cytotoxic T cells (CD8+) or helper T cells (CD4+) that coordinate immune responses.
• TCR requires antigen presentation by MHC molecules. CD4+ T cells recognize antigens presented by MHC class II, while CD8+ T cells recognize antigens presented by MHC Class I.

Antibodies

• Also called immunoglobulins, are a component of the immune system and are important for humoral immunity.
• They are generally considered important for humoral immunity. spanning Immune functions for many immune cells and overall pathogen elimination.

Immunoglobulin (IG) Molecule

• Human IGs contain four polypeptides: two identical light chains and two identical heavy chains linked by disulfide bonds.
• This gives the traditional Y-shaped formation.
• Heavy and light chains are subdivided into domains.
• Light chains can be either kappa (chromosome 2) or lambda (chromosome 22).
• Heavy chains can be mu, delta, gamma, epsilon, or alpha; these genetically different heavy chains are called isotypes.

IG Anatomical Features

• Different regions in the IG molecule provide different functional roles.
• IG molecules can be enzymatically cleaved into discrete fragments by either pepsin or papain.
• Papain cleaves heavy chains to form two identical fragments (Fab).
• Pepsin cleaves heavy chains to produce a single linked antigen binding fragment.
• A single IG contains identical light chains and identical heavy chains, variability of the antigen binding fragment ensures antibodies can recognize a broad repertoire of antigens.

IG Fragments

• Fab (antigen binding fragment) is the "business end" of the molecule and Pepsin produces two Fab fragments.
• Fc (constant fragment) facilitates the eventual isotype from the heavy chain and Pepsin digestion reduces the Fc fragment
• FD is the heavy chain portion of Fab while FD prime is a heavy chain produced by pepsin.
• Fab Prime is the linked Fab produced by pepsin cleavage.

Immunoglobulin Molecule - Hinge region

• Short, flexible segment between the Fab (antigen-binding) and Fc (constant) regions.
• Provides flexibility, enabling the two Fab regions to move independently, allowing the antibody to adjust its structure to bind to antigens.

B Cell Receptor components

• The B cell receptor has two heavy chains and two light chains and the region or fragment of the B cell receptor that binds the antigen is called the fragment antigen binding, or fab region.
• The Fab region is where the ends of the heavy chain and light chains meet, and there are two fab fragments on each B cell receptor.
• The remainder of the heavy chain makes up the constant region, or constant fragment region, also called FC. The two heavy chains are linked to one another by disulfide bonds, and each heavy chain is also linked to the light chain by a disulfide bond.
• Each BCR has two identical heavy and light chains, and as the B cell develops into a plasma cell, the B cell receptor gets treated as an antibody with the exact same antigen specificity. The heavy chain changes as the B cell develops. There are five major types of heavy chains which are immunoglobulins: IgM, IgD, IGT, I Ida and IGE.

BCR Fragments

• Variable (V), Diversity (D), and Joining (J) Segments undergo somatic recombination during B cell development contributing to the variable regions of the BCR and it provides diversity to the antigen-binding regions of the BCR, allowing recognition of a wide range of antigens.
• Variable (V) Regions: Located at the tips of the Y-shaped BCR structure, formed by the variable domains of the heavy and light chains; responsible for direct binding to antigens, leading to B cell activation.
• Constant (C) Regions located in the stem of the Y-shaped BCR structure, formed by the constant domains of the heavy and light chains; contribute to the overall structure and stability of the BCR.
• Transmembrane Region: Hydrophobic region anchoring the BCR to the B cell membrane. Secures the BCR in the cell membrane, allowing it to be expressed on the B cell surface.
• Cytoplasmic Tail Located inside the B cell; contains signaling motifs that become activated upon antigen binding, initiating intracellular signaling cascades.

TCR Fragments

• V,D, and J Segments undergo somatic recombination during T cell development contributing to the variable regions of the TCR; providing diversity to the antigen-binding regions of the TCR, allowing recognition of a wide range of antigens.
• V Regions Located at the tips of the TCR structure and recognize antigens presented by MHC molecules on the surface of antigen-presenting cells (APCs)
• C Regions Located in the stem of the TCR structure; contribute to the overall structure and stability of the TCR.
• Transmembrane Region: Hydrophobic region anchoring the TCR to the T cell membrane. Secures the TCR in the cell membrane, allowing it to be expressed on the T cell surface.
• Cytoplasmic Tail contains signaling motifs that become activated upon TCR engagement with antigen-MHC complexes, initiating intracellular signaling cascades.

Immunoglobulin Production

• Humans produce all five IG isotypes, B cells produce and display only one heavy chain isotype.
• Unstimulated B cells express both IgM and IgD.

IgD - Antigen Sensitivity

• IgD acts as a secondary receptor on naïve B cells, alongside IgM, to help in antigen recognition and immune tolerance.
• Dual expression allows immune system to fine-tune responses.
• IgD has higher antigen-binding flexibility than IgM.
• It is less resource-intensive than producing other isotypes like IgG or IgA.

IgG

• IgG is located on the surface or secreted. There exists four subclasses of IgG, and collectively makes up the greatest amount of IG molecule in the serum.
• Primary Immunoglobulin involved in forming soluble immune complexes, and is the most abundant antibody in circulation.
• IgG antibodies bind to self-antigens to form immune complexes. The deposition of IgG-containing immune complexes activates the complement system, recruits immune cells, and leads to inflammation and tissue damage.

IgM

• Found as a cell surface monomer or secreted pentamer. The first antibody to be formed during an initial immune response.

IgA

• IgA is found in a monomeric formation in the serum. However, the addition of a chain links to monomers forming a dimer, and is present in this formation at mucosal surfaces.

IgE

• IgE is present at relatively low levels in the serum and predominantly found on the surface of mast cells and basophils that have isotype specific receptors for recognition of IgE.

IgD

• IgD is almost exclusively found on the surface of B cells.

Antibody Effector Functions

• Functions result from interactions between heavy chain constant regions and other serum proteins or membrane receptors.
• Because effective functions result from interactions between heavy chain constant regions and other serum proteins or membrane receptors, not all antibody classes have the same functional properties

Immunoglobulin classes

• Immunoglobulin are classified on their heavy chain isotypes.
• Immunoglobulin A has Alpha heavy chain, IgD has delta heavy chain, IGP has epsilon heavy chain, IgG has gamma heavy chain, and IgM has new heavy chain.
• Light chain isotex, and we see there are two types of it, which can either be kappa or lambda chain, or all the immunoglobulins.
• IgG is most abundant of all, and it constitutes the 75 to 80% of total immunoglobulin
• IGT, IGE and IgG oppose in monomer form only, whereas IgM mostly oppose in pentamarket occur in monomo forms as well as it's expressed on plasma. We may not be telling both sides as a monomer.
• IGA is present in external secretions like breast milk, deals and Bucha, gastroint, respiratory Ethereum anymore, the IGA opposing two forms, IGA one and IGA two. The IGA is present in mono Marie form.
• IgG, which is memorable immunoglobulin, which is expressed by MarieTV B cells. It plays a role in activation and differentiation of B cell moving towards IgG antibody and is the only immunoglobulin that can pass through the placenta.
• Immunoglobulin M, which is present in membrane more B cells and activates complement system, that is the classical pathway.

Soluble vs. Insoluble Immune Complexes

• IgG antibodies
• Formed in circulation when antibodies bind to antigens.
• Typically involve IgG antibodies.
• Remain soluble in blood and other bodily fluids.
• Circulate freely in the bloodstream.
• Can deposit in tissues, leading to inflammation and tissue damage.
• Insoluble antibody complexes form larger insoluble complexes, often with IgM involvement and they are more prone to deposit in blood vessel walls or other tissues, leading to localized inflammation and damage

IgG - Mediated Disease

• Soluble immune complexes of IgG and self-antigens deposit in tissues, can deposit in various tissues, contributing to inflammation and damage.
• Examples: Joint pain, skin rashes, kidney involvement, and other systemic manifestations is Systemic Lupus Erythematosus (SLE).

IgM Mediated Diesase

• Insoluble immune complexes deposit in the glomeruli of the kidneys, leading to inflammation and impaired renal function.
• Examples: Acute glomerulonephritis, Hematuria, proteinuria, and hypertension.

Immunoglobulin (Ig) Classes and Subclasses

• IgM (Immunoglobulin M) has a Pentameric structure in its secreted form; Monomeric structure on the surface of B cells used to Agglutination and Activates the complement system.
• IgD (Immunoglobulin D) has a Monomeric structure and acts as a Functions as a membrane receptor for antigen recognition; Role in B cell activation and differentiation.
• IgG (Immunoglobulin G) has a Monomeric structure that provides long- Provides long-term immunity (secondary immune response), Opsonization of pathogens for phagocytosis and Activates the complement system.
• IgA (Immunoglobulin A) has Monomeric structure in circulation and Dimeric structure (joined by a J chain) Provides mucosal immunity and found Provides mucosal immunity in tears, saliva, colostrum, and other secretions.
• IgE (Immunoglobulin E) Monomeric structure involved Involved in allergic reactions; Controls parasitic infections and Binds to Fc receptors on mast cells

Immunoglobulins in Host Defenses

• Neutralization: Antibodies can neutralize toxins and viruses by binding to their active sites.
• Opsonization: Antibodies enhance phagocytosis by binding to pathogens.
• Complement Activation: Antibodies can activate the complement system.
• Agglutination: Antibodies promote the clumping of microbes, making it easier for phagocytes to capture and eliminate them.
• Mucosal Immunity: IgA provides protection at mucosal surfaces
• Allergic Reactions: IgE mediates allergic responses

Antigen

• molecule recognized by the immune system, primarily B and T cell lymphocytes
• Epitopes (antigenic determinants): Smallest part of an antigen recognized by lymphocytes.
• Antibodies are glycoproteins, and therefore can function as a potent immunogen to induce an antibody response and anti IG antibodies are viable tools to study femoral immune responses
• Antigenic determinants (epitopes) on antibodies are isotypic, callotypic, and pediatric.

Isotypic Determinants definition

• located on the constant region (Fc region) of the heavy chain of an immunoglobulin
• defining the class or subclass of the antibody.
• determined by the genetic sequence of the constant region of the heavy chains
• They help define the functional properties of the antibody: activating complements, binding to immune cell response receptors, and participation in the immune response.

Alltypic Deteminants definition

• Generated by the confirmation of the amino acid sequences of the heavy and light chain variable regions specific for each antigen.

Role of Epitopes in Immune Activation

• Recognition by B Cells, through B cell receptors binding
• Recognition by T Cells, as peptides presented on MHC molecules, leading to Activation and recruitment of other immune cells

Adjuvants

• They are not part of the antigen
• Act like a 'danger signal' to promote a stronger immune response

IgG vs IgM

• Soluble immune complex that binds antibodies to red blood cells do not experience cross linking, while insoluable ones experience cross-linking.

B-Cell vs T-Cell Epitopes

• Consists of both a linear and non linear sequences, almost always consist of a linear sequence of amino acid residues. They generally are located in the interior of a protein antigen on the cell surface, and located on the surface they lose their immunogenicity when an protein antigen is benatured by heat
• Immunodominant epitotes are detemined by the MHC molecules expressed by the individual
• The both generally arise from protiens
• May have multiple different epitotes on the same antigen
• Immuogenicity depends on the 3 demensional structure of an antigen

IGA Secretions Role

• Molecules in external secretions important for physical and chemical barriers to infection in the health and protection of the body, such as in breast milk, mucus, saliva and tears.

IGA Secretions Structure

• Secretory molecule consists of two IGA molecules joined together via J chain and secretory component.

Secretory IGA Function

• Serves a performs critical effector function at mucous membrane surfaces and main entry sites for most pathogenic microorganisms. It forms when dimeric IGA binds to a poly ID receptor on the basal lateral membrane of an epithelial cell, and is internalized by receptor mediated endocytosis
• Transport involves receptor mediated endocytosis of clatherin pits, vesicle transport across epithelial cell to the luminal/apical membrane, vesicle plasma membrane fusion, and enzymatic cleavage.

Secretory IgA Formation Stepwise

• Plasma Cells synthesize IgA then IgA molecules which is linked together through a J chain
• Dimeric IgA then bind to the poly-Ig receptor (pIgR)
• The receptor-IgA complex is endocytosed and transported across the cell
• At the apical surface then The poly-Ig receptor is cleaved enzymatically, and the secretory component (SC) of the pIgR is separated from the dimeric IgA
• Secretory IgA is released where it acts as a defense mechanism

Immunogens

• Immunogens promotes a specific and positive immune response
• Haptens are small antigens that are typically non immunogenic, bound to an immunogen can induce an immune response utilized to study tolerance
• Tolerogens are molecules that induce immune unresponsiveness

Immunogenicity and Antigenicity

• Immunogenicity refers to the ability of an immunogen to induce a humoral and or cell mediated immune response.
• Antigenicity refers to the degree to which an antigen is recognized by the immune system.

Burkitt Lymphoma Connection to Immunoglobulin Light Chains

• Is often linked to genetic rearrangements that involve the MYC gene, which results in the overexpression of the MYC protein, which drives uncontrolled cell division and contributes to the development of the lymphoma.