Inmunología I - 5 Clase: Generación de diversidad de los receptores BCR y TCR

Understanding the Diversity of BCR and TCR in Adaptive Immunity

Introduction to Adaptive Immune System

• The session begins with a focus on how diversity is generated in B cell receptors (BCR) and T cell receptors (TCR) within lymphocytes.

• Emphasis is placed on the adaptive immune system's ability to recognize pathogen components more specifically compared to innate immunity, which relies on danger signals.

Mechanisms Behind Receptor Diversity

• The adaptive immune system generates numerous receptor types that are similar yet distinct, allowing for recognition of a wide array of pathogens.

• A molecular understanding of the mechanisms at the DNA level that enable millions of clones with slight variations in recognition capabilities will be explored.

Overview of Key Receptors: Antibodies and TCR

• Two main receptors discussed are antibodies (part of B cells) and TCR, highlighting their roles during infections when activated by specific pathogen components.

• Most B cell clones exist as naive lymphocytes with membrane-bound antibodies until they encounter an antigen, leading to activation and differentiation into plasma cells that secrete antibodies.

Functions and Recognition Capabilities

• Antibodies can directly act on various antigens, recognizing diverse structures such as amino acid sequences, lipids, or carbohydrates regardless of size.

• In contrast, TCR remains anchored on the surface of T cells and recognizes peptide fragments presented by MHC molecules from infected cells. This process allows T cells to detect intracellular infections effectively.

Presentation Mechanism via MHC Molecules

• The presentation involves processing viral proteins into peptides ranging from 8 to 20 amino acids that bind to MHC molecules for recognition by TCR. This enables detection of infections at a cellular level.

• The importance of this mechanism lies in its ability to alert T cells about internal cellular conditions through peptide-MHC interactions during infections.

Conclusion: Similarities Between TCR and Antibodies

Understanding Antibody Structure and Function

Overview of Antibodies and T-Cell Receptors

• The discussion begins with a focus on antibody molecules, transitioning towards T-cell receptors (TCR) later in the presentation. The adaptive immune system can generate an immense variety of receptors.

Unique Recognition Sites of Antibodies

• Each antibody molecule possesses a unique recognition site formed by variable domains, specifically the heavy chain's variable domain (VH).

• The typical structure of antibodies resembles the letter "Y," consisting of a heavy chain and light chain, each containing variable domains.

Binding Specificity and Diversity

• The binding site, known as the paratope, is formed by the association of variable domains. This diversity allows for nearly any structure entering the body to be recognized by some paratope present.

• Emphasizes that antibody-antigen interactions must be specific, requiring complementary surfaces between antigens and antibody binding sites.

Epitope Recognition

• Discusses the relationship between paratopes and epitopes; where paratopes are antibody binding sites, epitopes are specific regions on antigens recognized by antibodies.

Structural Characteristics of Variable Domains

• Focus shifts to studying the primary structure of variable domains in antibodies, which consists of 110 amino acids with three highly variable regions known as CDR1, CDR2, and CDR3.

Folding into Functional Structures

• As these structures fold into secondary forms, CDR regions form loops within beta sheets that contribute to immunoglobulin domain stability through hydrogen bonding.

Formation of Antibody Binding Sites

• In tertiary structure formation, CDR regions come together spatially to create functional binding sites or paratopes for antigen interaction.

Clonal Diversity in Immune Response

• Highlights how adaptive immunity generates vast clonal diversity during lymphocyte development—both T-cells and B-cells produce numerous clones with unique receptors based on their variable domains.

Mechanism Behind Clonal Selection

• Each clone is phenotypically similar but differs at receptor level due to unique variations in their binding sites. This results in billions of distinct clones capable of recognizing various pathogens.

Avoiding Autoimmunity

• It’s crucial for the immune system to prevent autoreactive clones from forming that could lead to health issues.

Summary Insight on Immune Activation

Understanding Lymphocyte Cloning and Immune Response

The Development of Lymphocytes

• The process begins with lymphocytes that are similar yet distinct, evolving to produce more effective clones as needed.

• From birth, individuals develop the capacity to generate lymphocytes independently of infections, marking a crucial aspect of immune development.

• A diverse array of lymphocyte clones is generated randomly, preparing the body for various pathogens encountered throughout life.

Proliferation and Memory in Immune Response

• There is a continuous turnover of lymphocyte clones; they are constantly produced and eliminated based on need.

• Upon infection, specific pathogen molecules select particular clones for proliferation, leading to the differentiation into effector cells like antibody-secreting B cells and memory cells.

• Memory cells play a vital role in faster and more effective responses during reinfections by recalling previous encounters with pathogens.

Activation Mechanisms During Infection

• The innate immune system detects known pathogens first; it activates inflammation which is critical for initiating adaptive immune responses.

• Inflammation facilitates the movement of antigens from infected tissues to lymph nodes where further immune activation occurs.

Role of Dendritic Cells

• Dendritic cells serve as key connectors between innate and adaptive immunity by activating T lymphocytes upon antigen presentation.

• In lymph nodes, B lymphocytes interact with incoming antigens, setting the stage for their activation and subsequent response.

Diversity in Immune Repertoire

• The discussion raises questions about how such a vast repertoire (10^9 - 10^10 different proteins/antibodies) can be generated from a single genome line.

Understanding Immune Diversity

The Role of Lymphocytes in Immune Response

• The discussion begins with the question of how vaccines utilize the existing repertoire of lymphocytes, which are clones capable of recognizing foreign antigens.

• It is noted that most lymphocyte clones have a short lifespan and may not encounter any antigens during their lifetime, raising questions about the probability of activation upon infection or vaccination.

• A key inquiry is made regarding how the immune system generates an almost limitless diversity of clones from a single genome, suggesting potential mechanisms for this diversity.

Mechanisms Behind Lymphocyte Diversity

• The speaker proposes that millions of antibody genes could exist within the genome, leading to diverse receptor formations among lymphocytes.

• Each lymphocyte develops unique receptors through various combinations of genes during its formation, even before encountering an infection.

• There is a mention of genetic recombination processes that allow for different arrangements and combinations in receptor formation.

Genetic Recombination and Clonal Variation

• The conversation highlights that while there may be random combinations in receptor formation, multiple receptors can coexist within individual lymphocytes to enhance detection capabilities against infections.

• Each lymphocyte ultimately expresses a unique receptor due to somatic recombination occurring during development, ensuring clonal specificity despite shared genetic backgrounds.

Somatic Recombination Process

• Somatic recombination occurs exclusively during lymphocyte development and involves rearrangements in genes coding for variable domains in antibodies or T-cell receptors (TCR).

• This process results in new DNA configurations as segments are combined randomly from groups of similar gene segments found within the genome.

Generating Antibody Diversity

• As each clone develops, it combines gene segments through somatic recombination to create unique variable regions essential for antigen recognition.

• An example illustrates how one clone might combine specific gene segments differently than another clone, leading to distinct antigen-binding sites across various clones.

Recombination of DNA and Antibody Development

Overview of DNA Recombination

• Discussion on the role of DNA recombination in coding for antibodies, specifically focusing on heavy and light chains.

• Introduction to somatic recombination processes during B lymphocyte development.

Development of Lymphocytes

• Explanation of where B and T lymphocytes develop: B cells in bone marrow and T cells in the thymus.

• Emphasis on the orderly process of lymphocyte development through various cellular stages.

Key Stages in B Lymphocyte Development

• Identification of critical stages in B cell development where somatic recombination occurs, leading to antibody chain formation.

• Description of mature B lymphocytes expressing unique antibodies (IgD or IgM), highlighting their specificity.

Characteristics of Virgin B Lymphocytes

• Virgin B lymphocytes are described as mature but have not yet encountered specific antigens; they primarily express IgM or IgD.

• Mention that these virgin cells circulate through secondary lymphoid organs like lymph nodes and spleen.

Genetic Distribution for Antibody Chains

• Overview of gene distribution responsible for heavy and light chains in humans, including variations across species.

• Detailed explanation about gene segments (B, D, J segments) that contribute to variable domains necessary for antibody diversity.

Estimating Combinatorial Diversity

• Discussion on how combinatorial diversity is estimated based on available gene segment combinations (B, D, J).

Understanding the Role of B Segments in Antibody Diversity

The Contribution of B Segments to Variable Domains

• The B segments carry the majority of information for the variable domain, specifically CDR1, CDR2, and part of CDR3. The remaining information in CDR3 is derived from combinations with D and J segments.

• In light chains, similar patterns are observed where B segments contribute significantly to CDR1, CDR2, and part of CDR3. This indicates that variability in these regions is influenced by segment selection.

• CDR3 exhibits the highest variability among the complementarity-determining regions (CDRs). While CDR1 and CDR2 depend on selected B segments, their variability is limited compared to that of CDR3.

Estimating Antibody Diversity

• The diversity can be estimated based on known numbers of B, D, and J segments. For lambda genes alone, there are 120 possible combinations due to 30 potential B segments combined with four J segments.

• Similarly for kappa chains: 40 B segments can combine with five J segments resulting in 200 possible kappa chains. This leads to a total of 320 light chain combinations paired with approximately 6,000 heavy chain possibilities.

• By multiplying these combinations (320 light chains x 6,000 heavy chains), we arrive at an estimate of around two million unique antibody variants solely from these segment combinations.

Factors Influencing Antibody Diversity

• Despite estimating millions of potential antibodies from segment combinations alone, actual diversity reaches billions due to additional factors not yet discussed.

• Somatic recombination plays a crucial role in generating this diversity; however, molecular mechanisms behind it require further exploration through literature or detailed study materials provided later.

Mechanism Behind Somatic Recombination

• Two key recombinase enzymes—RAG1 and RAG2—are expressed during specific stages in the development of B and T lymphocytes. Their expression is critical for initiating somatic recombination processes.

• These recombinases act specifically within the BDJ gene region responsible for variable domains because conserved sequences exist within introns separating these segments that facilitate pairing during recombination events.

Process of DNA Repair During Recombination

• When loops form between paired sequences during recombination cuts made by RAG enzymes lead to excision. This results in DNA needing repair by cellular machinery which brings together D and J segments post-cutting.

• As D joins with J and similarly for light chains (B joining with D or J), this process generates new configurations contributing further to antibody diversity through random nucleotide addition or deletion during repair phases.

Conclusion on Combinatorial Diversity

DNA Repair Machinery and Its Role in Diversity

The Impact of Random Nucleotide Addition

• The process of DNA repair machinery introduces random nucleotide additions, contributing to immense diversity in segments that is difficult to measure due to its randomness.

Mutations and Their Consequences

• Random mutations can lead to the generation of truncated clones, which may not express functional heavy or light chains due to premature stop codons.

• While many mutations result in cell death from truncation, some are beneficial and allow for the survival of functional lymphocytes.

Enhancing Clonal Diversity

• There are two types of diversity: combinatorial diversity and junctional diversity, both crucial for increasing the initial number of clones significantly beyond 2 million.

• This process can potentially generate up to 10^9 or 10^10 clones circulating within the organism.

Receptor Generation Process

Stages of Receptor Development

• The receptor generation process is orderly; it does not occur simultaneously but progresses through distinct stages starting with pro-B cells rearranging heavy chain segments.

Allelic Rearrangement

• Each B cell has two genes for each chain (one on each chromosome), allowing for testing multiple rearrangements until a productive heavy chain is formed.

• If no functional heavy chain is produced after testing both alleles, the cell undergoes apoptosis.

Pre-B Cell Receptor Formation

• A pre-BCR forms when a successfully rearranged heavy chain associates with an invariant light chain, allowing functionality testing before further development.

Proliferation and Light Chain Rearrangement

Transitioning Through Stages

• Upon successful formation of a pre-BCR, the pro-B cell receives survival signals leading to proliferation while retaining the same heavy chain.

Light Chain Gene Rearrangement

• In subsequent stages as pre-B cells develop, they begin rearranging their light chain genes systematically across different alleles until a functional light chain is formed.

Allelic Exclusion Phenomenon

Ensuring Unique BCR Expression

• During this developmental process, only one allele remains active at a time due to allelic exclusion, ensuring that each B lymphocyte expresses a unique BCR on its membrane.

Biological Significance

Understanding B Lymphocyte Development and Antibody Response

Mechanisms of B Lymphocyte Selection

• A B lymphocyte membrane recognizes antigens through antibodies, which can lead to the secretion of non-selected antibodies that may be harmful, potentially causing autoimmune issues.

• During development, immature B cells test their B cell receptors (BCR) against self-antigens in the bone marrow; recognition of self-antigens triggers negative survival signals.

• If a B cell recognizes a self-antigen, it must undergo gene rearrangements. Failure to do so or inappropriate recognition leads to apoptosis.

Gene Rearrangement and Antibody Expression

• The germline DNA contains all segments necessary for heavy chain genes; during differentiation, a B lymphocyte rearranges its DNA segments (V-D-J recombination).

• Successful rearrangement results in gene expression for antibodies, producing processed mRNA with V-D-J segments linked to heavy chain genes.

• This process allows for the expression of different antibody classes (e.g., IgD and IgM), leading to naive B cells having both types on their membranes.

Activation and Differentiation of Mature B Cells

• Upon activation by an antigen, the first antibody secreted is typically IgM rather than IgD; the reason for this distinction remains unclear.

• The development process includes stages where mature B cells migrate peripherally and remain inactive until they encounter specific antigens.

Proliferation and Memory Formation

• Activated B cells proliferate significantly, generating progeny that can differentiate into plasma cells (antibody-secreting cells) or memory B cells crucial for reinfection responses.

• Plasma cells secrete antibodies dynamically throughout infection phases; this response evolves over time as more activated lymphocytes respond.

Changes in Antibody Response Over Time

• Following immunization with an antigen in laboratory settings, initial immune responses show predominance of low-affinity IgM antibodies within days.

• As time progresses post-infection or vaccination, there is a shift towards higher concentrations of high-affinity IgG antibodies replacing IgM dominance.

Understanding Antibody Affinity Maturation and Somatic Hypermutation

Overview of Changes in Lymph Nodes

• The discussion begins with changes occurring at the peripheral level, specifically in lymph nodes, rather than the bone marrow. These changes relate to antibody affinity maturation and class switching.

• Emphasis is placed on somatic recombination happening in the bone marrow during development, transitioning to peripheral processes involving circulating B lymphocytes.

Activation of B Lymphocytes

• Activated B lymphocytes undergo somatic hypermutation, which involves point mutations occurring exclusively in the variable regions of both light and heavy chains of antibodies.

• This process is unique to B lymphocytes that receive help from T lymphocytes, highlighting the importance of T-dependent responses for effective antibody production.

Mechanism of Somatic Hypermutation

• The mutation rate during somatic hypermutation is significantly higher than normal cellular mutation rates, affecting only variable gene regions while leaving constant regions intact.

• A key enzyme involved is activation-induced cytidine deaminase (AID), which facilitates conversion of cytosine to uracil within DNA sequences when a B cell is activated.

Role of AID in Mutation Process

• AID acts on "hotspot" regions within antibody genes characterized by specific nucleotide sequences (WRCY), leading to targeted mutations primarily in variable segments.

• The introduction of uracil creates mismatches during DNA replication, prompting repair mechanisms that can result in permanent mutations.

Repair Mechanisms Following Mutations

• When mismatches occur due to uracil incorporation, cellular repair machinery recognizes these errors and may introduce further mutations through various pathways.

• The repair process can lead to different outcomes: either correcting the mismatch or introducing new nucleotides that alter the original sequence permanently.

Understanding Somatic Hypermutation and Affinity Maturation

The Role of Somatic Hypermutation

• Somatic hypermutation involves the removal of uracil and adjacent nucleotides, requiring repair machinery to add new nucleotides based on the complementary strand. This process can lead to mutations.

Purpose of Mutations in Immune Response

• These mutations are utilized by the immune system to generate clones with higher affinity antibodies, enhancing their ability to bind effectively to antigens present in the body.

Affinity Maturation Process

• Affinity maturation occurs in specific sites within lymph nodes, where activated B cells proliferate after encountering an antigen, leading to a diverse population of B cells.

Germinal Centers Formation

• Activated B cells form germinal centers in lymph nodes, consisting of numerous identical progeny that differ slightly due to somatic hypermutations.

Selection for High-Affinity Cells

• A selection process occurs within germinal centers where only those B cells that improve their affinity for the antigen survive, while others undergo apoptosis due to low affinity.

Mechanisms Behind Antibody Class Switching

Changes Over Time in Antibody Production

• The maturation of antibody affinity changes over time alongside class switching events at the genetic level affecting antibody expression.

Genetic Structure of Antibodies

• The typical gene distribution for human heavy chains includes segments associated with IgM (mu), IgD (delta), and other classes like IgG (gamma), IgA (alpha), and IgE (epsilon).

Initial Antibody Expression

• Initially expressed antibodies are IgD and IgM; however, upon activation, a cell secretes primarily IgM before potentially undergoing class switching.

Class Switching Mechanism

• Class switching allows B cells to change from expressing IgM/IgD to other classes like IgG or IgA through DNA modifications involving recombination between gene segments.

Role of Activation-Induced Cytidine Deaminase (AID)

Processes in Germinal Center B Cells

Class Switching and Somatic Hypermutation

• Class switching and somatic hypermutation occur simultaneously within germinal center B cells, indicating a coordinated process.

• These two processes are genetically independent but happen concurrently at the same location, highlighting their interrelated nature during immune response.

• Activation-induced cytidine deaminase (AID) promotes double-strand breaks in DNA, facilitating the recombination necessary for class switching.

Mechanism of DNA Loop Formation

• Following DNA cuts, loops or "bucles" form through pairing of damaged DNA segments, similar to mechanisms seen in somatic recombination.

• The formed loops guide the association of variable segments (BDJ) with nearby constant gene segments (e.g., IgG, IgA), determining the type of antibody produced.

Summary of B Cell Development

• B cell development begins in the bone marrow where changes lead to receptor generation; peripheral activation occurs upon antigen exposure.

• Activated B cells differentiate into secretory cells while undergoing class switching and affinity maturation through somatic hypermutation.

Transitioning to T Cell Receptor Development

Similarities Between TCR and Antibody Structure

• The structure of T cell receptors (TCR), particularly alpha-beta TCR, shares similarities with antibodies, featuring constant and variable domains.

• TCR recognizes peptides presented by MHC molecules on antigen-presenting cells, necessitating recognition of both self and non-self components for activation.

Genetic Rearrangement in TCR Expression

• The genetic basis for TCR expression involves rearrangement similar to that seen in antibody production; specific gene segments are combined during this process.

• In humans, chromosome 14 contains various gene segments for alpha chains while chromosome 7 houses beta chain genes; these undergo productive rearrangements leading to functional TCR formation.

Distribution Variations Among Gene Segments

Understanding T Cell Development

Overview of T Cell Segments

• The transcript discusses various segments involved in T cell development, including segments B, G, and J, with a focus on the recombination process associated with specific constants (gamma).

Development in the Thymus

• T cell development occurs primarily in the thymus, which has distinct regions: the medulla and cortex. This structure is crucial for understanding how T cells mature.

Arrival of Thymocytes

• Thymocytes (immature T cells) enter the thymus from bone marrow but have not yet fully developed. They are still undergoing maturation processes.

Transition to Double Negative Thymocytes

• Upon entering the thymus, thymocytes migrate to the cortical area where they are classified as double negative (DN), indicating they have not yet committed to becoming CD4 or CD8 T cells.

Gene Rearrangement Process

• During their development, DN thymocytes undergo gene rearrangements starting with the beta chain. Successful formation of this chain leads to further maturation steps.

TCR Formation and Selection

Formation of Pre-TCR Complex

• The beta chain associates with an invariant surrogate alpha chain to form a pre-TCR complex. This complex plays a critical role in signaling and further development.

Inhibition of Other Rearrangements

• The presence of a functional pre-TCR inhibits rearrangements of gamma and delta chains, guiding thymocytes towards either gamma-delta or alpha-beta lineage decisions.

Proliferation After Productive Rearrangement

• If a productive rearrangement occurs (e.g., successful beta chain formation), DN thymocytes proliferate while maintaining identical beta chains among them.

Positive Selection Mechanisms

Testing by Antigen-Presenting Cells

• Once alpha-beta TCR is formed, it is tested against self-peptides presented by antigen-presenting cells within the thymus to ensure proper recognition capabilities.

Importance of MHC Recognition

• Positive selection aims to identify those thymocytes that can moderately recognize MHC molecules presenting self-peptides; this step is vital for future immune responses.

Differentiation into CD4 or CD8 Cells

• Depending on whether they recognize class I or class II MHC molecules during positive selection, thymocytes will differentiate into either CD8 or CD4 T cells respectively.

Negative Selection Processes

Migration to Medullary Region

• Following positive selection, single-positive thymocytes migrate back into the medullary region where they undergo further selection processes involving more diverse self-peptides presented by other APCs.

Elimination of Self-Reactive Clones

• This stage promotes elimination through apoptosis for those clones that strongly recognize self-peptides while allowing moderate recognizers to proliferate and migrate outwards into peripheral tissues.

Development and Tolerance of Lymphocytes

Overview of Lymphocyte Development

• The session concludes with a discussion on the origin of T lymphocytes, which develop in a specific area, and B lymphocytes, which originate from earlier developmental processes discussed in class.

• Emphasis is placed on understanding how receptor generation occurs simultaneously with lymphocyte development, highlighting the roles of B lymphocytes in bone marrow and T lymphocytes at the thymus.

• The instructor checks for any doubts regarding the material covered, indicating an interactive approach to ensure comprehension among participants.

Conclusion and Next Steps

• The session wraps up with gratitude expressed towards participants for their presence and engagement throughout the class.