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T-CELL DEVELOPMENT CENTRAL TOLERANCE ARPAD LANYI PhD The cellular organization of the thymus The proportion of the thymus that produces T-cells decreases with age DIFFERENTIATION OF T-CELLS IN THE THYMUS REGULATED T-CELL DIFFERENTIATION CD4-CD8- pro T-cell NO T –CELL RECEPTOR CD4+CD8+ preTCR cell preEpithelial T-cell SIGNALING RECEPTOR CD4+/CD8+ TCR naive T-cell ANTIGEN RECOGNIZING immature 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 signaled to divide and differentiate. After around a week, have lost stem-cell markers and have become thymocytes committed to the T-cell lineage (pro T-cell), as seen expression of the T-cell specific adhesion molecule CD2. cells are the cells that are by their 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 β-, α-, δ- and γ-chain loci Each germline TCR locus includes variable (V), joining (J) and constant (C) gene segments. TCR β and TCR δ loci also have D segments, like the Ig heavy chain locus. The basic rules of TCR rearrangement are identical to that of the BCR. δ gene segments are embedded within the a-chain locus. α-chain gene rearrangement results in the deletion of the δ-chain locus. V(D)J RECOMBINATION Recombination signal sequences: conserved hepta- and nonamer sequences CACAGTG; ACAAAAACC spacer regions: 12/23bp V(D)J recombinases Recognize RSSs and bring together two coding segments. RAG1 makes a nick: generates free 3’-OH and 5’-P Endonuclease. 3’-OH attacks phosphodiester bond on the Opens up thea hairpins at the coding ends. other strand forming Mutation of Artemis: T- aB-hairpin. NK+ SCID The blunt signal ends are ligated together and discarded. Double-stranded DNA repair enzyme. Activates Artemis. Mutation of DNA-PK: T- B- NK+ SCID Adds bases to broken DNA ends. Lymphoid-specific. 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) non–template-encoded nucleotides by TdT The y- and δ-chain loci contain fewer V gene segments, BUT during δ-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 δ-, γ- and β-chain genes leads to early commitment of some cells to the γ:δ T-cell lineage. δ- and γ-chain genes rearrange before β-chain and γ:δ receptor assembles. Signals through γ:δ TCR stop further rearrangement. γ:δ T-cells mature, leave the thymus and travel to other tissues via the blood. variable region (V) constant region (C) transmembrane region cytoplasmic tail γδ 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 δ-, γ- and β-chain genes leads to early commitment of some cells to the γ:δ T-cell lineage. δ- and γ-chain genes rearrange before β-chain and γ:δ receptor assembles. Signals through γ:δ TCR stop further rearrangement. γ:δ T-cells mature, leave the thymus and travel to other tissues via the blood. The more frequent outcome of the competition between the β-, y- and δchain genes is for a productive β-chain gene rearrangement to be made before both productive y- and δ-chain rearrangements occur. TCR β-CHAIN GENE REARRANGEMENT 1ST CHECKPOINT Rearrangement of a Vβ, a Dβ and a Jβ gene segment After translocation tofunctional the endoplasmic reticulum creates the V-domain exon. β-chain is tested for its capacity to bind to an invariant polypeptide called pTα, Unused V and J genes between the rearranged which acts as a surrogate α-chain. V and J genes are deleted. TCR β-CHAIN GENE REARRANGEMENT This possibility is not available to the immunoglobulin heavychain genes, because a nonproductive rearrangement deletes all the non-rearranged D segments. A nonproductively rearranged β-chain gene can also be rescued by a second rearrangement at the same locus. If a rearrangement at one β-chain locus is nonproductive, a thymocyte can attempt a rearrangement at the β-chain locus on the homologous chromosome. Thymocytes can make four attempts to rearrange the β-chain gene The potential for up to four β-chain gene rearrangements means that 80% of thymocytes make a productive rearrangement of the β-chain gene, compared with a 55% success rate for heavy-chain gene rearrangement by developing B cells. T-CELL DIFFERENTIATION Rearrangement of the δ-, γ- and β-chain genes leads to early commitment of some cells to the γ:δ T-cell lineage. δ- and γ-chain genes rearrange before β-chain and γ:δ receptor assembles. Signals through γ:δ TCR stop further rearrangement. γ:δ T-cells mature, leave the thymus and travel to other tissues via the blood. The more frequent outcome of the competition between the β- y- and δchain genes is for a productive β-chain gene rearrangement to be made before both productive y- and δ-chain rearrangements occur. If the β-chain binds to pTa, this heterodimer assembles with the CD3 complex and ζ-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 α, γ, and δ loci, but not to the β-chain locus. A minority of the doublepositive thymocytes give rise to additional γ:δ T-cells. Upon rearrangement of the α-chain locus, the δ-chain locus it contains is eliminated as part of an extrachromosomal circle. TCR α-CHAIN GENE SUCCESSIVE REARRANGEMENT 2ND CHECKPOINT After translocation to the endoplasmic reticulum α-chain is tested for its capacity to bind the β-chain and assemble a T-cell receptor. A Vα gene segment rearranges to a Jα 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 δ-, γ- and β-chain genes leads to early commitment of some cells to the γ:δ T-cell lineage. δ- and γ-chain genes rearrange before β-chain and γ:δ receptor assembles. Signals through γ:δ TCR stop further rearrangement. γ:δ T-cells mature, leave the thymus and travel to other tissues via the blood. The more frequent outcome of the competition between the β- y- and δchain genes is for a productive β-chain gene rearrangement to be made before both productive y- and δ-chain rearrangements occur. If the β-chain binds to pTa, this heterodimer assembles with the CD3 complex and ζ-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 α, γ, and δ loci, but not to the β-chain locus. A minority of the doublepositive thymocytes give rise to additional γ:δ T-cells. Upon rearrangement of the α-chain locus, the δ-chain locus it contains is eliminated as part of an extrachromosomal circle. Productive α-chain gene rearrangements produce double-positive CD4 CD8 α:β T-cells. This ends the early stage of T-cell development. GENE EXPRESSION THROUGH THE STAGES OF α:β 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 β selection CD4 T-cells: survival Effector CD4 T-cells: Th2 polarization Only a small fraction of T-cells mature into functional T-cells POSITIVE SELECTION -Occurs in the cortex, requires thymic epithelial cells. -αβ double-positive thymocytes must recognize selfMHC. 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 TCRα 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 SIDE BY SIDE VIEW OF T-AND B-CELL DEVELOPMENT Nemazee Nature Reviews Immunology 6, 728–740 (October 2006) | doi:10.1038/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 Immune response IMMUNOLOGICAL TOLERANCE Immunological tolerance 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. T-cells with high affinity TCR towards the self MHC/self peptide complex are eliminated, but clones with intermediate affinity survive. NEGATIVE SELECTION Elimination of potentially CENTRAL TOLERANCE KEYWORD: AFFINITY of T-cells in THE the thymus autoreactive clones 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. Peripheral tolerance: Elimination of „fugitive” or altered clones is an important role for regulatory T-cells. SUMMARY 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 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- CANDIDIASISECTODERMAL 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 by studying a rare immunodeficiency. • https:///jimneydandme.wordp ress.com/james-story THE END V(D)J RECOMBINATION 1. 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-1 is 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 β-CHAIN GENE REARRANGEMENT Rearrangement of a Vβ, a Dβ and a Jβ gene segment creates the functional V-domain exon. Unused V and J genes between the rearranged V and J genes are deleted. 1ST CHECKPOINT After translocation to the endoplasmic reticulum β-chain is tested for its capacity to bind to an invariant polypeptide called pTα, which acts as a surrogate α-chain. SUCCESSIVE TCR α-CHAIN GENE REARRANGEMENT 2ND CHECKPOINT After translocation to the endoplasmic reticulum α-chain is tested for its capacity to bind the β-chain and assemble a T-cell receptor. A Vα gene segment rearranges to a Jα 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 CENTRAL TOLERANCE Elimination of potentially autoreactive clones 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