T cell development, Generation of receptor repertoire diversity Flashcards
Where do T cells come from?
T cells originate from hematopoietic stem cells (HSCs) in the bone marrow
HSCs differentiate into common lymphoid progenitors (CLPs) which have the potential to differentiate into various lymphoid cell lineages, including T cells, B cells, and natural killer (NK) cells.
After commitment to the T cell lineage, the progenitors migrate from the bone marrow to the thymus, where they undergo a series of developmental stages to differentiate into mature naive T cells
Describe the main steps and processes T cells follow to become mature naive cells
The main steps and processes T cells follow to become mature naive cells involve a combination of cellular proliferation, differentiation, and selection processes within the thymus. These processes include:
1) Migration to the thymus: After commitment to the T cell lineage, T cell progenitors leave the bone marrow and enter the thymus via the bloodstream. This migration is guided by chemokines and adhesion molecules that facilitate the entry of these cells into the thymus.
2) Commitment to the T cell lineage: Upon arrival in the thymus, T cell progenitors commit to the T cell lineage by upregulating T cell-specific genes and downregulating genes related to other cell lineages. This lineage commitment is mediated by Notch1 signalling
3) T cell receptor (TCR) gene rearrangement: T cells undergo V(D)J recombination, a process that involves the rearrangement and recombination of gene segments encoding the TCR. This process generates a diverse repertoire of TCRs capable of recognising a wide range of antigens. TCR gene rearrangement occurs in a stepwise manner, with the β (or δ) chain rearranging first, followed by the α (or γ) chain.
4) Positive selection: T cells that successfully rearrange their TCR genes and are capable of interacting with self-major histocompatibility complex (MHC) molecules on cortical thymic epithelial cells undergo positive selection. This process ensures that only T cells that can recognise self-MHC molecules, which are essential for presenting antigens to T cells, are allowed to survive and mature.
5) Negative selection: T cells that have a high affinity for self-peptide-MHC complexes are eliminated through a process called negative selection mediated by medullary thymic epithelial cells. This process helps prevent autoimmune responses by removing T cells that could potentially react with self-antigens.
6) Lineage commitment: After positive and negative selection, T cells differentiate into one of two major subtypes: CD4+ helper T cells or CD8+ cytotoxic T cells. This decision is based on the interaction of the TCR with MHC class II or class I molecules, respectively. CD4+ T cells primarily help coordinate immune responses, while CD8+ T cells directly target and kill infected or abnormal cells.
7) Exit from the thymus: Mature naive T cells that have successfully completed their development in the thymus exit into the peripheral circulation, where they can participate in immune responses against foreign pathogens or abnormal cells.
Name a few important cell surface receptors, transcription factors, and soluble cytokines that mediate the early steps of T cell development
Cell Surface Receptors
- Notch1: Interactions between Notch1 on thymocytes (immature T cells) play a critical role in T cell lineage commitment. Notch1 signalling, initiated through the interaction with Delta-like ligands on thymic epithelial cells, is critical for the commitment of hematopoietic progenitor cells to the T cell lineage. It helps suppress the developmental pathways of other lymphoid and myeloid lineages, thus ensuring the progenitor cells differentiate into T cells.
- IL-7 Receptor (IL-7R): consists of an IL-7Rα chain and a common gamma chain (γc). It binds to the cytokine IL-7 = survival and proliferation of early thymocytes by JAK/STAT signalling pathway leading to the expression of anti-apoptotic proteins and cell cycle regulator essential for the survival and proliferation of early T cell progenitors.
- Pre-T Cell Receptor (pre-TCR): The pre-TCR is a heterodimer consisting of the TCRβ and pre-Tα chains. It is expressed on the surface of immature T cells and plays a crucial role in the selection and expansion of T cells expressing functional TCRβ chains.
Transcription Factors
- T-cell factor 1 (TCF-1) and GATA-binding protein 3 (GATA-3): These transcription factors regulate the expression of genes that are crucial for early T-cell development, including Notch1 and the IL-7R.
- B-cell lymphoma/leukaemia 11b (Bcl11b): This transcription factor is required to commit T cell progenitors to the T cell lineage and to repress alternative lineage potentials.
- Runt-related transcription factor 1 (Runx1): Runx1 regulates the expression of CD4 and CD8, the coreceptors for MHC class II and class I molecules, respectively.
Soluble Cytokines
- Interleukin-7 (IL-7): Produced by thymic stromal cells, IL-7 is crucial for the survival and proliferation of early T cell progenitors. It interacts with the IL-7R on the surface of these cells to promote their development.
- Stem Cell Factor (SCF): SCF binds to the c-Kit receptor on early T cell progenitors and synergizes with IL-7 to promote the survival and proliferation of these cells.
- FLT3 Ligand: This cytokine binds to the FLT3 receptor on T cell progenitors and plays a role in their proliferation and differentiation. It often works in combination with other cytokines like IL-7 and SCF.
Identify the regions of the thymus where T cell development occurs as well as the structural cells involved in the process
Thymic Cortex: the outermost region of the thymus where T cell development begins, here, T cells are known as cortex immature thymocytes or T cell precursors, migrated from the bone marrow:
- T cell precursors begin expressing both CD4 and CD8 co-receptors, becoming double-positive (DP) thymocytes
- The T cell receptor (TCR) undergoes rearrangement. Successful rearrangement leads to the expression of the pre-TCR, which signals the thymocytes to proliferate and transition to the DP stage
- DP thymocytes undergo positive selection, assess the ability of the TCR to recognise and bind self-MHC molecules presented by cortical thymic epithelial cells (cTECs). Thymocytes with TCRs capable of binding self-MHC molecules with low affinity survive, those that cannot undergo apoptosis
Thymic Medulla: Thymocytes that survive positive selection migrate to the inner region of the thymus known as the medulla:
- Undergo negative selection, thymocytes with high affinity for self-peptides presented by medullary thymic epithelial cells (mTECs) and dendritic cells are eliminated. This process aid in preventing autoimmunity
- Thymocytes that survive negative selection down-regulate either CD4 or CD8, becoming single positive (SP) T cells
Structural cells involved in T cell development:
- Thymic Epithelial Cells, cTECs = positive selection, mTECs = negative selection
- Thymic Dendritic Cells → present self-peptides to developing thymocytes and contribute to negative selection
- Macrophages → clearing apoptotic thymocytes that fail positive or negative selection
Describe the structure of the T cell receptor
Composition of TCR:
- The TCR is a heterodimeric transmembrane protein, composed of two different polypeptide chains: either an alpha (α) chain and a beta (β) chain or a gamma (γ) chain and a delta (δ) chain.
- In most T cells, the TCR is an αβ receptor, while γδ TCRs are found on a smaller subset of T cells.
- Each chain is composed of a variable (V) region and a constant (C) region
Variable (V) Region:
- The variable region of the TCR is responsible for antigen recognition.
- The V region of each chain consists of three highly variable complementarity-determining regions (CDRs).
- Among these, CDR3 is the most variable and plays the most significant role in antigen recognition.
- The V region of the α chain pairs with the V region of the β chain (or the γ chain pairs with the δ chain in γδ T cells) to form the antigen-binding site of the TCR.
Constant (C) Region:
- The constant region of the TCR is less variable and is involved in anchoring the TCR to the cell membrane and communicating with other components of the T cell signalling machinery.
- The C region of each chain includes a transmembrane segment that anchors the TCR in the lipid bilayer of the cell membrane and a short cytoplasmic tail. However, the cytoplasmic tail of the TCR is too short to transduce signals into the cell.
Associated Molecules, TCR associates with several other proteins to form the TCR complex:
- CD3 complex (composed of CD3γ, CD3δ, and two CD3ε chains) and the ζ-chain homodimer.
- Both the CD3 complex and the ζ-chain have long cytoplasmic tails containing immunoreceptor tyrosine-based activation motifs (ITAMs), which are critical for signal transduction. Upon TCR engagement with an antigen-MHC complex, the ITAMs are phosphorylated, activating various intracellular signalling pathways that ultimately activate T cells.
Describe the structure of the MHC class II
MHC 2 are molecules that are expressed on the surface of antigen-presenting cells (APC) and present antigenic peptides to CD4+ T cells, primarily helper T cells
Overall Structure:
- MHC class 2 molecule is a heterodimer composed of 2 non-identical protein chains: the α and the β chains. Both chains are transmembrane glycoproteins containing carbohydrate moieties and traverse the cell membrane
Extracellular Domain:
- Each chain of the MHC class II molecule is made up of two extracellular domains: the α1 and α2 domains form the alpha chain, and the β1 and β2 domains form the beta chain
- The α1 and β1 domains together create a groove on the extracellular surface of the molecule for binding processed antigenic peptides, the groove is open-ended, unlike MHC class 1 which are closed-ended
- The binding of peptides to the MHC 2 is facilitated by multiple pockets within the binding groove. These pockets are made up of polymorphic residues, which vary widely among MHC 2 molecules and contribute to the enormous diversity of peptides that can be presented
- The α2 and β2 domains form an immunoglobulin-like structure beneath the α1 and β1 domains, providing structural support to the molecule
Transmembrane domain:
- The α and β chains each contain a hydrophobic transmembrane domain that anchors the MHC 2 molecule within the lipid bilayer of the cell membrane
- The location of the MHC 2 on the cell membrane allows it to interact directly with CD4+ T cells in the immune response
Cytoplasmic domain:
- The α and β chains have cytoplasmic tails that protrude into the interior of the cell
- These tails contain sorting signals that direct the intracellular transport of the MHC class 2 molecules, interacting with cytoskeletons.
Invariant chain and CLIP:
- In the ER, MHC 2 associate with a protein called the invariant chain, which: 1) prevents premature binding of peptides to MHC class 2, 2) guides MHC class 2 to the endosomal pathway where antigens are processed.
- The invariant chain is later degraded, leaving a small fragment called CLIP (Class 2-associated invariant chain peptide) in the peptide-binding groove. The exchange of CLIP for antigenic peptide is facilitated by HLA-DM in humans
Explain the consequences of the structural relationship between the TCR and the MHC
- Specificity: TCR has a specific binding site that interacts with both the peptide and the MHC molecule
- Diversity: The TCR repertoire is incredibly diverse, due to gene rearrangement processes that occur during T cell development
- MHC Restriction: T cell, through its TCR recognises a peptide antigen only when it is presented by a particular MHC molecule, CD8+ T cells only recognise MHC class 1 molecules and CD4+ T cells only recognise MHC class 2 molecules, crucial for the appropriate activation and response of these T cells
- Self-tolerance: During T cell development in the thymus, T cells that have a high affinity for self-peptides presented by self-MHC molecules are eliminated in a process called negative selection. This process helps prevent autoimmunity by eliminating T cells that respond to self-antigens
- Effector functions: Interaction of TCR with the peptide-MHC complex leads to T cell activation, CD8+ T cells can become cytotoxic T cells that kill infected or cancerous cells, while CD4+ T cells can differentiate into various types of helper T cells that assist other immune responses
Describe the genetic processes that produce the TCR
Occurs in the thymus
1) Gene Segments and Rearrangement:
- The TCR genes cannot be transcribed and translated directly into a functional receptor in the germline DNA
- Instead, they are composed of separate gene segments. For TCR α and δ chains, these segments are called Variable (V), Diversity (D, δ chain only), and Joining (J) segments. For the TCR β and γ chains, there are V, D (β chain only), J, and Constant (C) segments
- The process of TCR gene rearrangement is directed by recombination signal sequences (RSSs) located adjacent to each gene segment
- The RSSs are recognised by a group of enzymes collectively known as the V(D)J recombinase, which includes the proteins RAG1 and RAG2, this enzyme complex brings together two selected gene segments, cleaves their DNA, then ligate them to form a continuous sequence that can be transcribed
- This process first occurs for the TCR β, γ, and δ chains (D-J rearrangement followed by V-DJ rearrangement), and if the β chain successfully rearranges, it then occurs for the α chain (V-J rearrangement). The result is a unique combination of gene segments for each chain of the TCR, contributing to the diversity of antigen recognition
2) Junctional Diversity:
- Further diversity is introduced at the junctions between the rearranged gene segments by the addition or deletion of nucleotides
- This is facilitated by the enzyme terminal deoxynucleotidyl transferase (TdT), which randomly adds extra nucleotides (N regions) at the junctions
- The precise trimming and filling of the junction can also result in the loss or gain of nucleotides (P regions), adding to the variability
3) Combinatorial Diversity
- Each T cell expresses a unique TCR, and this is achieved by the combination of a unique α chain with a unique β chain (or γ with δ for γδ T cells). This combinatorial association of different chains greatly expands the diversity of the TCR repertoire
4) Allelic Exclusion
- Ensures each T cell produces a TCR of a single specificity, whereby successful rearrangement of one TCR chain (β or α) on one chromosome halts further rearrangement attempts on the other chromosome. This ensures that each T cell expresses TCRs with a single antigenic specificity
Identify the stages of early T cell development where TCR rearrangements take place
1) Double negative stage (CD4- & CD8-):
- Earliest stage of T cell development in the thymus where the cells at the stage are called thymocytes
- The double negative stage is further divided into DN1 (CD44+ CD25-), DN2 (CD44+ CD25+), DN3 (CD44- CD25+), and DN4 (CD44- CD25-) subsets based on the expression of other cell surface markers
- During the DN2 and DN3 stages, the TCRβ, γ, and δ gene segments are rearranged. If the rearrangement is successful, the cells express a pre-TCR complex (TCRβ chain associated with pre-Tα and CD3) on the surface and progress to the DN4 stage
2) Double positive stage (CD4+ CD8+)
- After successful β chain rearrangement, cells undergo rapid proliferation and become double positive (DP) thymocytes, expressing both CD4 and CD8 co-receptors
- During this stage, the rearrangement of the TCRα gene occurs. If the rearrangement is successful, the α chain pairs with the β chain and the cells express a complete αβ TCR on their surface
3) Single positive stage (CD4+ CD8- or CD4- CD8+)
- DP thymocytes expressing a functional TCR undergo positive selection based on their ability to recognise self-peptide-MHC complexes.
- Thymocytes that successfully undergo positive selection downregulate either CD4 or CD8 to become single positive (SP) thymocytes.
- If TCR binds to MHC class I molecules, the cell will downregulate CD4 and maintain CD8 expression, becoming a CD8+ cytotoxic T cell. If the TCR binds to MHC class II molecules, the cell will downregulate CD8 and maintain CD4 expression, becoming a CD4+ helper T cell
- These cells then undergo negative selection to eliminate self-reactive T cells where those that bind self-antigen-MHC complexes with too high affinity undergo apoptosis before they are released as mature T cells
Explain the concept of allelic exclusion
- a key process in the development of lymphocytes (both B cells and T cells) that ensures monospecificity – the production of a receptor that recognizes only one specific antigen.
- Each T cell has two sets (or alleles) of TCR genes, one from each parent. The principle of allelic exclusion stipulates that only one of these two sets of genes is rearranged to produce a functional TCR in each T cell
- TCRβ locus rearranges first, during the Double Negative (DN) stage of development. This rearrangement process is stochastic, starting on one chromosome randomly. If a functional TCRβ chain is produced, it pairs with the pre-Tα chain (a surrogate alpha chain) to form the pre-TCR. This sends a signal halting further rearrangement at the TCRβ locus on the second allele, thus implementing allelic exclusion at the TCRβ locus
- The TCRα locus, in contrast, rearranges later during the Double Positive (DP) stage. Once a functional TCRα chain is produced, it pairs with the already produced TCRβ chain, forming a complete and functional αβ TCR complex. The formation of this complex sends a signal to halt further TCRα rearrangement. As the TCRα locus rearrangement process is a bit more complicated and involves deletion of previously rearranged TCRα gene segments
- Also aids in preventing the development of auto-reactive T cells, as the process of positive and negative selection in the thymus tests the self-reactivity of only one unique TCR
Apply your prior knowledge of Flow Cytometry to identify the different stages of T cell development
In the context of T cell development, different stages can be distinguished by the presence or absence of certain cell surface proteins, such as CD4, CD8, and CD3, along with the TCR
1) Double negative (DN) stage:
- Absence of CD4 and CD8 expression, thus in the quadrant of negative for both in the flow cytometry plot
- Further subdivided: DN1 = CD44+ CD25-, DN2 = CD44+ CD25+, DN3 = CD44- CD25+, DN4: CD44- CD25-
2) Double positive stage:
- Following successful β-selection, the T cells begin to express both CD4 and CD8, becoming double positive (DP). In a flow cytometry plot, these cells would appear in the quadrant positive for both CD4 and CD8
3) Single Positive (SP) Stage:
- On a flow cytometry plot, these cells would appear in the quadrants showing only CD4 or CD8 positivity
4) Mature T cells:
- You can identify mature CD4+ and CD8+ T cells by their high expression of TCR and CD3.
