T-CELL DEVELOPMENT CENTRAL TOLERANCE
ARPAD LANYI PhD The cellular organization of the thymus
The figure shows the detailed structure of a lobus.The left panel shows a microscopic image of a stained lobus section, and the right panel shows the same structures schematically.Large number of immature thymocytes (blue) is found in the darkly staining cortex, together with the network of cortical epithelial cells (orange).The medulla contains mature thymocytes, medullar epithelial cells (dark orange), dendritic cells and macrophages (yellow).It also contains the Hassal's corpuscules. The proportion of the thymus that produces T-cells decreases with age
Starting at birth, the T-cell producing tissue of the thymus is gradually replaced by fatty tissue. This process is called the involution of the thymus. The graph shows the percentage of thymic tissue that is still producing T-cells at different ages. The micrograph in panel a shows a section through the thymus of a 3-day-old infant; the micrograph in panel b shows a section through the thymus from a 70-year-old person for comparison. Tissue is stained with hematoxylin and eosin (red and blue). Magnification x 20. Micrographs courtesy of Yasodha Natkunam. DIFFERENTIATION OF T-CELLS
IN THE THYMUS REGULATED T-CELL DIFFERENTIATION ANTIGEN RECOGNIZING RECEPTOR
CD4+CD8+preTCR CD4+/CD8+ TCR CD4-CD8- naive T-cell pre T-cell Epithelial cell ANTIGEN RECOGNIZING RECEPTOR pro T-cell immature T-cell SIGNALING RECEPTOR NO T CELL RECEPTOR T-CELL DIFFERENTIATION
T-cell precursors that enter the thymus express the hematopoietic stem-cell marker CD34 and the adhesion molecule CD44, but none of the characteristic markers of the T-cell lineage (CD2, CD3, CD5). Upon interaction with thymic stromal cells, the progenitor cells are signaled to divide and differentiate. After around a week, the cells have lost stem-cell markers and have become thymocytes that are committed to the T-cell lineage (pro T-cell), as seen by their expression of the T-cell specific adhesion molecule CD2. Commitment to the T-cell lineage is driven by the receptor Notch 1. Notch 1 on the thymocyte binds to its ligand on thymic epithelium. This interaction induces a protease to cleave the intracellular domain from the plasma membrane. The soluble intracellular domain is translocated to the nucleus, where it turns on the expression of genes essential for T-cell development by removing repressive transcription factors and recruiting co-activating transcription factors. T-CELL DIFFERENTIATION
Commitment to the T-cell lineage changes receptor expression Cells committed to the T-cell lineage express CD2, CD5, CD1a and IL7-receptor, but do not express T-cell receptor, CD4 or CD8 (double negative thymocytes). Lack of IL7 signaling (IL7 or IL7R) stalls early T-cell development. SCIDs. Cells are beginning to rearrange the TCR genes. TCR -, -, - and -chain loci
Each germline TCR locus includes variable (V), joining (J) and constant (C) gene segments. TCR and TCR loci also have D segments, like the Ig heavy chain locus. The basic rules of TCR rearrangement are identical to that of the BCR. gene segments are embedded within the a-chain locus. -chain gene rearrangement results in the deletion of the -chain locus. In all cells with the exception of matured lymphocytes TCR (and immunoglobulin) genes are in germline configuration. Transcription of these genes is possible only after the gene rearrangement. V(D)J recombination occurs only during lymphocyte development. V(D)J RECOMBINATION spacer regions: 12/23bp
Recombination signal sequences: conserved hepta- and nonamer sequences CACAGTG; ACAAAAACC V(D)J recombinases Recognize RSSs and bring together two coding segments. RAG1 makes a nick: generates free 3-OH and 5-P Endonuclease. Opens up the hairpins at the coding ends. Mutation of Artemis: T- B- NK+SCID Double-stranded DNA repair enzyme. Activates Artemis. Mutation of DNA-PK: T- B- NK+SCID Lymphoid specific . Expressed mainly in the G0 and G1 stages. Inactivated in proliferating cells. Rag-1is enzymatically active only when complexed with Rag-2. Mutation of RAG enzymes Omen syndrome, T- B- NK+ SCID 3-OH attacks a phosphodiester bond on the other strand forming a hairpin. The blunt signal ends are ligated together and discarded. Adds bases to broken DNA ends. Lymphoid-specific. Combinatorial diversity
V(D)J RECOMBINATION Combinatorial diversity V(D)J rearrangement brings together multiple germline gene segments that may combine randomly, and different combinations produce different antigen receptors. Junctional diversity The largest contribution to the diversity of antigen receptors is made by the removal or addition of nucleotides at the junctions of the V and D, D and J, or V and J segments. P nucleotides: nucleotides added to the asymmetrically cleaved hairpin ends N nucleotides: random added (up to 20) nontemplate-encoded nucleotides by TdT The y- and -chain loci contain fewer V gene segments, BUT during -gene rearrangement two D segments can be incorporated into the final gene sequence. increase in junctional diversity (extra N nucleotides between the two D segments) (+more combinations) T-CELL DIFFERENTIATION
Rearrangement of the -, - and -chain genes leads to early commitment of some cells to the : T-cell lineage. - and -chain genes rearrange before -chain and : receptor assembles. Signals through: TCR stop further rearrangement. : T-cells mature, leave the thymus and travel to other tissues via the blood. variable region (V) constant region (C) transmembrane region cytoplasmic tail T-cells MHC-independent, CD1c and CD1d dependent. Double megative.
Comprise about 1-5% of the T-cells found in the circulation, but can be the dominant (up to 50%) T-cell population in epithelial tissue. A population that is expanded in intra- (Mycobacterium tuberculosis and Listeria monocytogenes) and extracellular infections (Borrelia burgdorferi) and certain disease states such as celiac disease. T-CELL DIFFERENTIATION
Rearrangement of the -, - and -chain genes leads to early commitment of some cells to the : T-cell lineage. - and -chain genes rearrange before -chain and : receptor assembles. Signals through: TCR stop further rearrangement. : T-cells mature, leave the thymus and travel to other tissues via the blood. The more frequent outcome of the competition between the -, y- and -chain genes is for a productive -chain gene rearrangement to be made before both productive y- and -chain rearrangements occur. TCR -CHAIN GENE REARRANGEMENT
1ST CHECKPOINT Rearrangement of a V,a D and a J gene segmentcreates the functional V-domain exon. After translocation to the endoplasmic reticulum -chain is tested for its capacity to bind to an invariant polypeptide called pT, which acts as a surrogate -chain. Unused V and J genes between the rearranged V and Jgenes are deleted. Thymocytes can make four attempts to rearrange the -chain gene
TCR -CHAIN GENE REARRANGEMENT This possibility is not available to the immunoglobulin heavy-chain genes, because a nonproductive rearrangement deletes all the non-rearranged D segments. A nonproductively rearranged-chain gene can also be rescued by a second rearrangement at the same locus. If a rearrangement at one -chain locus is nonproductive, a thymocyte can attempt a rearrangement at the -chain locus on the homologous chromosome. Thymocytes can make four attempts to rearrange the -chain gene The potential for up to four -chain gene rearrangements means that 80% of thymocytes make a productive rearrangement of the -chain gene, compared with a 55% success rate for heavy-chain gene rearrangement by developing B cells. T-CELL DIFFERENTIATION
Rearrangement of the -, - and -chain genes leads to early commitment of some cells to the : T-cell lineage. - and -chain genes rearrange before -chain and : receptor assembles. Signals through: TCR stop further rearrangement. : T-cells mature, leave the thymus and travel to other tissues via the blood. The more frequent outcome of the competition between the - y- and -chain genes is for a productive -chain gene rearrangement to be made before both productive y- and -chain rearrangements occur. If the -chain binds to pTa, this heterodimer assembles with the CD3 complex and -chain to form the pre-T-cell receptor. Pre-TCR is sufficient for signaling and there is no requirement for binding a ligand.Pre-TCR induce pre T-cell to stop gene rearrangement (suppressing RAG1/2), proliferate and express CD4 and CD8 co-receptors (double-positive thymocytes). At this stage the recombination machinery is reactivated and targeted to the , , and loci, but not to the -chain locus. A minority of the double-positive thymocytes give rise to additional : T-cells. Upon rearrangement of the -chain locus, the -chain locus it contains is eliminated as part of an extrachromosomal circle. After translocation to the endoplasmic reticulum
TCR -CHAIN GENE REARRANGEMENT SUCCESSIVE 2ND CHECKPOINT After translocation to the endoplasmic reticulum -chain is tested for its capacity to bind the -chain and assemble a T-cell receptor. A V gene segment rearranges to a J gene segment to create a functional exon encoding the V domain. It continues until either a productive rearrangement occurs or the supply of V and J gene segments is exhausted, whereupon the cell dies (like Ig light chain). T-CELL DIFFERENTIATION
Rearrangement of the -, - and -chain genes leads to early commitment of some cells to the : T-cell lineage. - and -chain genes rearrange before -chain and : receptor assembles. Signals through: TCR stop further rearrangement. : T-cells mature, leave the thymus and travel to other tissues via the blood. The more frequent outcome of the competition between the - y- and -chain genes is for a productive -chain gene rearrangement to be made before both productive y- and -chain rearrangements occur. If the -chain binds to pTa, this heterodimer assembles with the CD3 complex and -chain to form the pre-T -cell receptor. Pre-TCR is sufficient for signaling and there is no requirement for binding a ligand.Pre-TCR induce pre T-cell to stop gene rearrangement (suppressing RAG1/2). The cells proliferate and express CD4 and CD8 co-receptors (double-positive thymocytes). At this stage the recombination machinery is reactivated and targeted to the , , and loci, but not to the -chain locus. A minority of the double-positive thymocytes give rise to additional : T-cells. Upon rearrangement of the -chain locus, the -chain locus it contains is eliminated as part of an extrachromosomal circle. Productive -chain gene rearrangements produce double-positive CD4 CD8 : T-cells. This ends the early stage of T-cell development. GENE EXPRESSION THROUGH THE STAGES OF : T-CELL DEVELOPMENT
Signals from the pre-T-cell receptor depend on the expression of the co-receptors CD4 and CD8, the signaling complex CD3, the tyrosine kinase ZAP-70, which is involved in relaying signals from the receptor, and the tyrosine kinase Lck, which is involved in signaling from the co-receptors. Ikaros regulates Notch target gene expression. Th-POK is required for the development of single-positive CD4 T-cells from double-positive thymocytes. Uncommitted progenitors: survival Committed thymocytes: optimal selection CD4 T-cells: survival Effector CD4 T-cells: Th2 polarization POSITIVE SELECTION THE KEYWORD: RECOGNITION
Only a small fraction of T-cells mature into functional T-cells POSITIVE SELECTION -Occurs in the cortex, requires thymic epithelial cells. double-positive thymocytes must recognize self-MHC. T-cells expressing TCRs that can bind to the self MHC/self-peptide complex on the surface of cortical epithelial cells will survive, but the others will die due to the lack of survival signals (death by neglect). Selection continues until a successful rearrangement on the TCR locus occurs (3-4 days). Ca. 2% of thymocytes survive!! Positive selection --- results in clones that are reactive to SELF MHC. BASIS OF MHC RESTRICTION!!! THE KEYWORD: RECOGNITION Positive selection of double positive (dp) T-cells also directs CD4 and CD8 single positive (sp) T-cell commitment THYMIC EPITHELIAL CELLS ARE MHCI/MHCII POSITIVE! BARE LYMPHOCYTE SYNDROME (BLS) Lack of MHC class I no CD8+ T-cells Lack of MHC class II no CD4+ T-cells T-AND B-CELL DEVELOPMENT
SIDE BY SIDE VIEW OF T-AND B-CELL DEVELOPMENT Common lymphoid progenitors (CLPs) give rise to both B-cell and T-cell lineages, but the development of B-cells and T-cells occurs separately, with B-cells developing in the bone marrow (bottom) and T-cells in the thymus (top). In progenitor (pro)-B-cells and progenitor (pro)-T-cells, the first antigen-receptor chain locus undergoes V(D)J recombination, generating the immunoglobulin heavy (IgH) chain in B-cells and the -chain of the T-cell receptor (TCR) in T-cells. (In addition, the genes that encode TCR and TCR recombine in pro-T-cells, sometimes giving rise to T-cells.) The first receptor chain then associates with the surrogate second receptor chain 5VpreB in precursor (pre)-B-cells, and the precursor (pre)-TCR -chain (pT) in pre-T-cells yielding a pre-B-cell receptor (pre-BCR) or pre-TCR complex, signaling through which mediates proliferation and developmental progression. Next, lymphocytes stop dividing and recombine the genes that encode the second receptor chain, generating immunoglobulin light (IgL) chain in B-cells and TCR in T-cells. In the main pathway of T-cell development (top), continued rearrangement at the TCRA locus often occurs because of autoreactivity or lack of positive selection, creating either a non-functional rearrangement (denoted by the reverse arrow) or an edited receptor with a new TCR. Positive selection ultimately stops gene rearrangements and promotes the loss of either CD4 or CD8. In B-cell development (bottom), successful recombination of the locus that encodes the IgL chain leads to cell-surface BCR expression on immature B cells. If the BCR is bound by autoantigen, these cells continue V(D)J recombination, resulting in receptor editing, particularly at loci that encode the IgL chain. Innocuous (non-self-reactive) receptors promote positive selection, and these B cells are subsequently released into the periphery. Ig:IgH chain with constant region. Nemazee Nature Reviews Immunology 6, 728740 (October 2006) | doi: /nri1939 The response of the immune system
to the stimuli of the outer and inner environment Environment Immune system Tolerance Self Non-self Dangerous Pathogenic The response of the immune system to the stimuli of the outer and inner environment. Antigens that are not harmful for the body will be tolerated by the immune system: examples are the molecules of our food or the body's own proteins. Pathogens that pose a danger will generate an immune response.The "black box" represents the immune system processing the stimuli from the environment. Immune response IMMUNOLOGICAL TOLERANCE Immunological tolerance Unlike immunosuppresion.
Definition: Unresponsiveness to a given antigen induced by the interaction of that antigen with the lymphocytes. ANTIGEN SPECIFIC!!! Unlike immunosuppresion. Why is this important? All individuals are tolerant to their own antigens (self tolerance). Failure of self tolerance results in autoimmunity. Terapeutic potential: Treat autoimmune diseases, allergic reaction or even tissue rejection. NEGATIVE SELECTION of T-cells in the thymus Elimination of potentially
T-cells with high affinity TCR towards the self MHC/self peptide complex are eliminated, but clones with intermediate affinity survive. NEGATIVE SELECTION of T-cells in the thymus Elimination of potentially autoreactive clones THE KEYWORD: AFFINITY CENTRAL TOLERANCE Development of central T-cell tolerance is an integral part of T-cell differentiation. T-cells incapable of interacting with self-MHC/self-antigen complexes are eliminated during positive selection, whereas clones with intermediate or high affinity towards the same complexes will survive and become single-positive T-cells. On the other hand, autoreactive clones are eliminated during negative selection by dendritic cells and other cells at the corticomedullary boundary of the thymus. T-cells with high affinity TCR towards the self MHC/self peptide complex are destroyed (negative selection, deletion), but clones with intermediate affinity survive. A percentage of self-reactive T-cells will survive the negative selection process, and differentiate into regulatory T-cells, an interesting cell type that have high affinity TCRs, bordering at negative selection, and have important roles in maintaning peripheral tolerance (discussed later). Immunological Tolerance and Autoimmunity : Self-Nonself Discrimination in the Immune System and Its Failure Abbas, Abul K., MBBS, Basic Immunology: Functions and Disorders of the Immune System, Chapter 9, A percentage of self-reactive T-cells that have high affinity TCRs, bordering negative selection will survive the negative selection process and differentiate into regulatory T-cells. Central and peripheral tolerance to self antigens
Central tolerance: Elimination of self-reactive clones. BUT!!! Some T-cell clones escape. Central and peripheral tolerance to self antigens. Central tolerance: Immature lymphocytes specific for self antigens may encounter these antigens in the generative (central) lymphoid organs and are deleted; B lymphocytes change their specificity (receptor editing); and some T lymphocytes develop into regulatory T-cells. Some self-reactive lymphocytes may complete their maturation and enter peripheral tissues. Peripheral tolerance: Mature self-reactive lymphocytes may be inactivated or deleted by encounter with self antigens in peripheral tissues, or suppressed by regulatory T-cells. Immunological Tolerance and Autoimmunity : Self-Nonself Discrimination in the Immune System and Its Failure Abbas, Abul K., MBBS, Basic Immunology: Functions and Disorders of the Immune System, Chapter 9, Peripheral tolerance: Elimination of fugitive or altered clones is an important role for regulatory T-cells. SUMMARY THE QUESTION: THE ANSWER:
CENTRAL T-CELL TOLERANCE IS SURPRISINGLY EFFECTIVE. MEDIATED MAINLY BY NEGATIVE SELECTION. THE QUESTION: HOW CAN TISSUE-RESTRICTED ANTIGENS BE EXPRESSED IN MEDULLARY THYMIC EPITHELIAL CELLS? THE ANSWER: AUTOIMMUN REGULATOR (AIRE)
A transcription factor expressed in the medulla of the thymus and induces expression of many tissue-specific genes DEFICIENCY IN ESTABLISHING CENTRAL T-CELL TOLERANCE
AIRE DEFICIENCY DEFICIENCY IN ESTABLISHING CENTRAL T-CELL TOLERANCE Lack of proper negative selection allows too many self reactive T-cell clones to leave the thymus AUTOIMMUNE POLYENDOCRINOPATHY- CANDIDIASIS-ECTODERMAL DYSTROPHY (APECED) Rare disease, but more frequently seen in inbred populations Finnish, Iranian Jews and in the island of Sardine SYMPTOMS OF APECED Anti-Th17 specific antibodies!!!!!
Role of Th17 discovered bystudying a rareimmunodeficiency. https:///jimneydandme.wordp ress.com/james-story THE END V(D)J RECOMBINATION Synapsis:
V(D)J recombinases recognizes recombination signal sequences (conserved hepta- and nonamer sequences flanking the V,D,J segments; spacer regions: 12/23bp). Recombination-activating gene 1-2 (Rag-1 and Rag-2): lymphoid specific expressed mainly in the G0 and G1 stages inactivated in proliferating cells Rag-1is enzymatically active only when complexed with Rag-2. mutation of RAG enzymes Omen syndrome, T- B- NK+ SCID Two selected coding segments and their adjacent RSSs are brought together by a chromosomal looping event. 2. Cleavage: Rag-1 makes a nick (on one strand) between the coding end and the heptamer. The released 3 OH of the coding end attacks a phosphodiester bond on the other strand, forming a covalent hairpin. The signal end (including the heptamer and the rest of the RSS) does not form a hairpin and is generated as a blunt double-stranded DNA terminus that undergoes no further processing. 3. Hairpin opening and end-processing: The broken coding ends are modified by the addition or removal of bases, and thus greater diversity is generated. Artemis: endonuclease, opens up the hairpins at the coding ends mutation of Artemis: T- B-NK+SCID Terminal deoxynucleotidyl transferase (TdT) lymphoid-specific adds bases to broken DNA ends 4. Joining: The broken coding ends as well as the signal ends are brought together and ligated. Double-stranded break repair process: nonhomologous end joining. DNA-dependent protein kinase (DNA-PK) double-stranded DNA repair enzyme activates Artemis mutation of DNA-PK: T- B-NK+SCID Ligation of the processed broken ends is mediated by DNA ligase IV and XRCC4. TCR -CHAIN GENE REARRANGEMENT
Rearrangement of a V,a D and a J gene segmentcreates the functional V-domain exon. Unused V and J genes between the rearranged V and Jgenes are deleted. 1ST CHECKPOINT After translocation to the endoplasmic reticulum -chain is tested for its capacity to bind to an invariant polypeptide called pT, which acts as a surrogate -chain. After translocation to the endoplasmic reticulum
SUCCESSIVE TCR -CHAIN GENE REARRANGEMENT 2ND CHECKPOINT After translocation to the endoplasmic reticulum -chain is tested for its capacity to bind the -chain and assemble a T-cell receptor. A V gene segment rearranges to a J gene segment to create a functional exon encoding the V domain. It continues until either a productive rearrangement occurs or the supply of V and J gene segments is exhausted, whereupon the cell dies (like Ig light chain). NEGATIVE SELECTION of T-cells in the thymus Elimination of potentially
autoreactive clones CENTRAL TOLERANCE T-cells with high affinity TCR towards the self MHC/self peptide complex are eliminated, but clones with intermediate affinity survive. A percentage of self-reactive T-cells that have high affinity TCRs, bordering at negative selection will survive the negative selection process and differentiate into regulatory T-cells. THE KEYWORD: AFFINITY