Patho 4.25 B-Cell and T-Cell Receptor Diversity and Development

B-Cell and T-Cell Receptor Diversity and Development

Introduction to Lymphocyte Receptors

• Adaptive immune cells (B-cells and T-cells) possess diverse lymphocyte receptors.

• These receptors must balance diversity (to recognize many antigens) with constant features (to trigger responses).

• Receptor development is integral to B and T lymphocyte maturation from stem cells in the bone marrow.

B-Cell Receptor/Antibody Overview

• B-cell receptors (BCRs) contribute to the immune system by binding to antigens.

• The process ensures that useful receptors are produced to respond to various pathogens.

• Lymphocytes have all necessary receptors before antigen exposure, a product of lymphocyte development.

• Antigen exposure improves receptor quality and quantity in B-cells.

Somatic Recombination

• T and B lymphocytes undergo somatic recombination, unique among somatic cells after birth.

• Immunoglobulin genes include:

  • Heavy chain.

  • Light chains: kappa and lambda.

• T-cell receptor genes include: alpha and beta chains.

• Gene segments include:

  • Variable (V) region (responsible for antigen binding).

  • Diversity (D) region.

  • Joining (J) segment.

Receptor Diversity Mechanisms

Combinatorial Diversity

• Recombination of V, D, and J segments creates diversity.

• B-cell receptors (immunoglobulin): ~3 million possibilities.

• T-cell receptors: ~6 million possibilities.

Junctional Diversity

• Nucleotides are added or subtracted at gene segment junctions.

• This alters the outcome of gene combinations.

• B-cell receptors: ~10^{11} possibilities (100 billion).

• T-cell receptors: ~10^{16} possibilities (10 quadrillion).

Checkpoints for Receptor Utility

• Not all receptor combinations are useful; some may bind too strongly.

• Checkpoints ensure receptor utility and prevent autoimmunity.

• Receptors are assessed for binding strength and potential for self-attack.

VDJ Recombination Ingredients

• Variability in antigen binding is balanced by invariable intracellular signaling.

• Receptors are bound to invariant molecules for intracellular signal transduction.

• This complex recognizes variable antigen binding and transmits signals.

Antigen Recognition and Signal Transduction

• Antigen binding leads to signal transduction.

• Variability in polypeptides allows for antigen recognition.

• Conserved polypeptides facilitate signal transduction.

• B-cell receptors:

  • Bind antigen.

  • Invariant chains (Igα and Igβ) transduce signals.

• T-cell receptors:

  • Require processed protein/peptide presented by MHC.

  • CD3 and zeta molecules transduce signals.

• Both receptor types use multiple polypeptides for signal transduction.

Comparison of B-Cell and T-Cell Receptors

Signaling Requirements

• Signaling occurs when adjacent antigen receptors cross-link by binding antigens.

• Cross-linking brings intracellular signaling molecules together.

• Enzymatic interactions (e.g., phosphorylation) initiate signal transduction.

• Outcome: gene activation through transcription.

• Antibodies: membrane-bound B-cell receptors or secreted forms (immunoglobulins).

• T-cell receptors: always membrane-bound.

B-Cell Receptor Structure and Function

• Antibody molecule/B-cell receptor/immunoglobulin is Y-shaped.

• Heterodimeric and glycosylated.

• Antibodies found in serum.

• B-cell receptors are bound to the plasma membrane.

• Antibodies bind foreign pathogens with high specificity.

• Adaptive system: antibodies are soluble (serum) or membrane-bound (BCRs).

Antibody Structure

• Four polypeptide chains:

  • Two heavy chains (H): ~50 kDa each.

    • One variable region.

    • Three or four constant domains (determine effector function and class).

  • Two light chains: ~25 kDa each.

    • One variable region.

    • One constant domain.

    • Kappa or lambda light chains (only one type expressed per B-cell receptor/antibody).

• Disulfide bonds link chains together.

• Two identical antigen-binding sites are formed by the variable regions of the heavy and light chains.

Antibody Regions

• Y-shaped with three equal-sized globular regions connected by the hinge region (flexible).

• Arms: light chains plus the amino terminus of heavy chains.

• Variable domains (VH and VL) at the amino termini bind antigen.

• Constant domains at the carboxy termini.

Proteolytic Fragments

• FAB fragment (fragment antigen-binding):

  • Contains the whole light chain.

  • The variable region of the heavy chain.

  • The first constant region of the heavy chain.

  • Binds antigen.

• FC fragment (fragment crystalline):

  • Remaining constant domains from heavy chain, where effector functions occur.

  • The hinge region links the FAB and FC regions.

Antibody Classes (Isotypes)

Antibody Classes and Their Properties

Antigen-Antibody Binding

• B-cell receptors and antibodies can bind various antigens, including macromolecules and small chemicals.

• Binding involves reversible non-covalent interactions (hydrogen bonds, charge).

• Only a small section of the antigen (epitope or antigenic determinant) binds.

• Epitopes can be linear (continuous) or conformational (discontinuous).

Affinity and Avidity

Affinity

• Strength of binding between the antibody surface and the antigen epitope.

• Expressed as the dissociation constant (Kd).

• Lower Kd = higher affinity.

• Initial Kd: micro to nanomolar concentrations.

Affinity Maturation

• Increase in antigen-binding strength over time in B-cells (not T-cells).

• Improves affinity to picomolar or femtomolar concentrations.

Avidity

• Total strength of binding in an antibody molecule.

• Always stronger than affinity for B-cell receptors because of multiple binding sites.

Cross-Reactivity

• Antibodies produced against one antigen may bind other structurally similar epitopes.

Hybridomas and Monoclonal Antibodies

Hybridomas

• Fused cells between a B-cell (producing a specific antibody) and myeloma cells (cancerous plasma cells).

• Myeloma cells provide immortality.

Monoclonal Antibodies

• Antibodies from a single clone of a desired specificity.

• Used as therapeutic agents (suffix "-mab").

Production

• Mouse is exposed to antigen.

• B-cells from the spleen are fused with myeloma cells.

• Fused cells are selected based on enzyme expression.

• Serial dilution to isolate single cells producing the desired antibody.

Examples of Monoclonal Antibodies and Their Targets

• Anti-TNF-alpha: blocks inflammation.

• Anti-IgE: blocks allergic responses.

• Anti-PD-1: cancer treatment.

• Anti-VEGF: cancer treatment.

Lymphocyte Development Principles

Properties of Mature Cells

• Gradually acquired in a stepwise process with progressively limited potential.

• Cell environment (bone marrow, thymus) provides developmental signals.

Survival Regulation

• Regulated through antigen receptors as they develop.

• Strong signals to immature lymphocytes lead to cell death or receptor rearrangement.

• Absence of signal also leads to apoptosis.

Selection Process

• Appropriate signaling through lymphocyte receptors leads to survival.

• Receptor must not be too strong or too weak.

Three Processes Involved in Lymphocyte Development

Proliferation

• Immature cells proliferate.

Antigen Receptor Gene Expression

• Appropriate signals commit cells to the lymphoid lineage.

• Failure to express appropriate antigen receptor leads to apoptosis.

Selection

• Pre-B or pre-T cell receptor takes the receptor out to test through binding.

• Immature B or T-cell expresses a fully expressed B or T-cell receptor.

Selection and Apoptosis

• If too strong, it's negatively selected.

• If just right, it's positively selected.

• Receptor editing in B cells.

• Ultimately, if those receptors don't work, the cell will die via apoptosis.

Early Lymphocyte Development Steps

Proliferation

• Initial proliferation is dependent upon IL-7 to maintain cells.

Antigen Receptor Expression

• Survival depends on how well the antigen receptors respond.

Selection

• Positive selection (T cells: MHC plus a peptide; B cells: useful receptors)

• Negative selection (high affinity for self-antigen = apoptosis)

Introduction to Lymphocyte Receptors

Adaptive immune cells, specifically B-cells and T-cells, exhibit remarkable diversity in their lymphocyte receptors, which play a critical role in the immune response. These receptors must strike a balance between a wide range of variability (which enables the recognition of myriad antigens) and certain consistent features (which are necessary to effectively trigger immune responses). The development of these receptors is not just a trivial aspect; it is a fundamental step in the maturation process of B and T lymphocytes originating from stem cells located in the bone marrow.

B-Cell Receptor/Antibody Overview

B-cell receptors (BCRs) provide essential contributions to the immune system by specifically binding to antigens. This binding process ensures that the repertoire of receptors produced is finely tuned to respond to various pathogens encountered throughout an individual's life. Remarkably, lymphocytes are pre-equipped with the necessary receptors before any exposure to specific antigens, a feat accomplished through intricate lymphocyte development. Upon encountering antigens, the quality and quantity of B-cell receptors are significantly enhanced, optimizing the immune response.

Somatic Recombination

A defining characteristic of T and B lymphocytes is their ability to undergo somatic recombination, a unique process that allows for genetic rearrangement, and is distinct from other somatic cells postnatally. Immunoglobulin genes consist of:

• Heavy chain genes.

• Light chain genes, which include kappa and lambda variants.
  T-cell receptor genes are composed of:

• Alpha and beta chain genes.
  Within these genes, specific segments known as:

• Variable (V) regions, which are responsible for antigen binding,

• Diversity (D) regions,

• Joining (J) segments, play crucial roles in receptor formation.

Receptor Diversity Mechanisms

1. Combinatorial Diversity
   The process of recombination involving V, D, and J segments creates substantial diversity in lymphocyte receptors. B-cell receptors can generate approximately 3 million unique possibilities, while T-cell receptors can yield an even broader spectrum of around 6 million unique combinations.

2. Junctional Diversity
   In addition to combinatorial diversity, there is further diversity generated at the junctions of gene segments where nucleotides can be either added or deleted. This variability allows for an astonishing potential of about 10^{11} (100 billion) different B-cell receptor combinations and a staggering 10^{16} (10 quadrillion) combinations for T-cell receptors.

Checkpoints for Receptor Utility

It is vital to recognize that not every receptor combination is beneficial for immune responses; some may lead to excessive binding or even self-reactivity. Thus, various checkpoints exist to assess the utility of receptors and to mitigate the risk of autoimmunity. These assessments involve rigorous evaluations of binding strength and the potential risk of self-attack, ensuring that only suitable receptors are allowed to mature.

VDJ Recombination Ingredients

The balance between variability in antigen binding and consistent intracellular signaling is facilitated by invariant molecules involved in signal transduction within the cells. These invariant receptors recognize diversified antigen binding and collaborate with signaling proteins to relay necessary signals for lymphocyte activation.

Antigen Recognition and Signal Transduction

When antigens bind to their respective receptors, this triggers a fundamental process known as signal transduction. This mechanism is critical for activating the immune response. Variability in polypeptides contributes significantly to the recognition of a wide array of antigens, while conserved polypeptides ensure the effective transduction of these signals. In B-cells, receptors bind antigens and are assisted by invariant chains (such as Igα and Igβ) that help in transmitting signals, whereas T-cell receptors depend on processed protein or peptide presented by Major Histocompatibility Complex (MHC) molecules, facilitated by CD3 and zeta molecules for signal transduction. Both receptor types utilize multiple polypeptides for effective communication and signal initiation.

Comparison of B-Cell and T-Cell Receptors

Signaling Requirements

Signaling within the immune system initiates when neighboring antigen receptors undergo cross-linking upon binding to antigens. This cross-linking effect brings intracellular signaling molecules into close proximity, triggering enzymatic interactions such as phosphorylation, which initiate the signal transduction cascade crucial for gene activation through transcription. Antibodies may exist in two forms: membrane-bound as B-cell receptors or soluble as secreted immunoglobulins, whereas T-cell receptors remain membrane-bound throughout their function.

B-Cell Receptor Structure and Function

The structural configuration of an antibody molecule (also referred to as B-cell receptor or immunoglobulin) is typically Y-shaped, characterized by both heterodimeric and glycosylated features. Antibodies circulate in serum and serve to bind to foreign pathogens with high specificity due to their intricate structure. The adaptive immune system employs antibodies, which can either be soluble in the serum or membrane-bound on the B-cells as BCRs.

Antibody Structure

An antibody comprises a total of four polypeptide chains:

• Two heavy chains (H), each approximately 50 kDa, featuring a variable region and three or four constant domains that influence the effector function and immunoglobulin class.

• Two light chains, about 25 kDa each, consist of one variable region and one constant domain, with either kappa or lambda light chains (note: only one type expressed per BCR/antibody).
  Disulfide bonds serve to link the chains together, forming two identical antigen-binding sites from the variable regions of both heavy and light chains.

Antibody Regions

The Y-shaped structure features three equal-sized globular regions, which are connected by a flexible hinge region. The arms consist of light chains along with the amino terminus of the heavy chains. The variable domains located at the amino termini are responsible for antigen binding, while constant domains are found at the carboxy termini, contributing to the overall stability of the antibody.

Proteolytic Fragments

1. FAB fragment (Fragment Antigen-Binding):

   • Comprises the entire light chain,

   • The variable region of the heavy chain,

   • The first constant domain of the heavy chain,

   • Specifically binds to antigens.

2. FC fragment (Fragment Crystalline):

   • Contains the remaining constant domains from the heavy chain,

   • This domain is where most effector functions occur.

   • A hinge region links the FAB and FC regions, enabling functional versatility.

Antibody Classes (Isotypes)

Antibody Classes and Their Properties

Antigen-Antibody Binding

B-cell receptors and antibodies exhibit the ability to bind a diverse range of antigens, encompassing both macromolecules and smaller chemical structures. The binding mechanism involves non-covalent interactions, primarily reversible interactions such as hydrogen bonds and electrostatic attractions, ensuring high specificity in the recognition process. It is noteworthy that only a minor section of the antigen, known as the epitope or antigenic determinant, is involved in this binding process. Epitopes can be classified into two categories: linear (continuous) epitopes comprised of consecutive amino acids, and conformational (discontinuous) epitopes, which require the native structure for binding.

Affinity and Avidity

1. Affinity:

   • Refers to the strength of the binding interaction between the antibody surface and the antigen epitope, quantified as the dissociation constant (Kd). A lower Kd signifies a higher affinity, typically indicating more effective binding dynamics at micro to nanomolar concentrations.

2. Affinity Maturation:

   • This process involves a systematic increase in antigen-binding strength in B-cells, enhancing their affinity to reach picomolar or femtomolar concentrations, which is not seen in T-cells.

3. Avidity:

   • Describes the overall strength of binding across the entire antibody molecule, which inherently is always stronger than affinity measurements alone due to the presence of multiple antigen-binding sites on B-cell receptors.

Cross-Reactivity

An important aspect of antibody specificity is cross-reactivity, wherein antibodies produced against a specific antigen may also exhibit binding to similar epitopes found on different antigens, highlighting the complexity and adaptability of the immune response.

Hybridomas and Monoclonal Antibodies

Hybridomas:
These are unique cell lines generated from the fusion of a B-cell, which is producing a specific antibody, with a myeloma cell, a type of cancerous plasma cell. The resultant myeloma cells contribute to the immortal character of the hybridoma, allowing for continuous antibody production.

Monoclonal Antibodies:
These antibodies originate from a single clone of cells exhibiting a specific binding affinity and are widely utilized as therapeutic agents, often denoted by the suffix “-mab.” They play a pivotal role in modern medicine and therapy.

Production Process:
The generation of monoclonal antibodies involves several steps:

1. Exposure of a mouse to a particular antigen.

2. Harvesting B-cells from the spleen of the immunized mouse.

3. Fusion of these B-cells with myeloma cells to create hybridomas.

4. Selection based on enzyme expression, identifying those cells producing the desired antibody.

5. Serial dilution of cultures to isolate single cells capable of producing the desired antibody in sufficient quantity.

Examples of Monoclonal Antibodies and Their Targets:

• Anti-TNF-alpha: effective in blocking inflammation processes.

• Anti-IgE: utilized to inhibit allergic responses.

• Anti-PD-1: plays a role in cancer treatment by blocking pathways that inhibit T-cell activity.

• Anti-VEGF: targets vascular endothelial growth factor for cancer therapeutics, inhibiting tumor growth by blocking blood supply.

Lymphocyte Development Principles

1. Properties of Mature Cells:
   The characteristics of mature lymphocytes develop progressively through a stepwise process, during which their potential is gradually limited. The surrounding environment, specifically within the bone marrow and thymus, provides essential developmental signals that shape lymphocyte maturation and function.

2. Survival Regulation:
   Survival of these immune cells is intricately regulated through signals conveyed by antigen receptors during their development. Immature lymphocytes receiving robust signaling cues may undergo cell death or receptor rearrangement, while a lack of an adequate signal can trigger apoptosis.

3. Selection Process:
   The correct signaling via lymphocyte receptors directly influences cellular survival. Optimal signaling requires that receptors exhibit neither excessive affinity (which could result in autoimmunity) nor insufficient strength (which could impede function). This selection process is crucial for maintaining an effective but self-tolerant immune system.

Three Processes Involved in Lymphocyte Development

1. Proliferation:
   Immature lymphocytes undergo extensive proliferation, a process that can be driven by interleukin-7 (IL-7) to sustain cell populations in their developmental stages.

2. Antigen Receptor Gene Expression:
   Developmental signals provide a commitment for cells to adhere to the lymphoid lineage. Any failure to express the necessary antigen receptor leads these cells to undergo apoptosis.

3. Selection:
   Immature B or T-cells are subjected to a selection phase where pre-B or pre-T-cell receptors are expressed to verify utility through binding tests. Successful receptors undergo positive selection, while those that bind too tightly are negatively selected. B-cells can further undergo receptor editing, and if all checks fail, the cell ultimately undergoes apoptosis.

Early Lymphocyte Development Steps

1. Proliferation:
   Initial proliferation of lymphocytes heavily relies on IL-7, a critical cytokine that maintains cell viability and promotes survival.

2. Antigen Receptor Expression:
   The capacity for survival is directly linked to the responsiveness of developing lymphocytes to the signals provided by their antigen receptors.

3. Selection:
   Positive selection ensures that T-cells recognize MHC along with a peptide, and B-cells express receptors that are functional. Negative selection screens out those receptors with high affinity for self-antigen, leading to apoptosis and fine-tuning the immune response.