2 Questions for the next 2 lectures: How do you generate a diverse T cell populationwith functional TCR rearrangements?How do you generate a T cell population that isself-MHC restricted?How do you ensure that those diverse T cellreceptors are not-self reactive?How do you coordinate lineage specification withMHC specificity and coreceptor expression?- vs. T cell- CD4 vs. CD8
3 a key regulator of the immune system T lymphocyte:a key regulator of the immune system
4 T LymphocytesArise from stem cells resident in the bone marrow and migrate to the thymus which serves as an indoctrination center where thymocytes must learn to distinguish self from nonselfThymocytes that cannot make this induction are eliminated, those that can may further differentiate, mature, and graduate as T lymphocytes and enter the circulation
5 Origin, generation and differentiation of T cells
6 T Cell DevelopmentT cell progenitors migrate from bone marrow and seed thymus.T cell progenitors undergo differentiation to CD4, CD8 and NKT cells in thymus.Mature CD4 and CD8 T cells circulate between blood and lymphoid tissues until they meet antigens presented on dendritic cells in lymphoid tissues.T cells further undergo maturation to become functional memory or effector T cells in LT
7 Thymic involution: Human thymus is fully developed before birth and increases in size until puberty. It then progressively shrinks during adult life.Most thymectomized adults have no problem in T cell immunity because they have enough memory T cells in the periphery, and these T cells are long-lived.Figure 5-2
8 T Cell FunctionsCollectively, T cells display a number of diverse functions:- They often function to initiate, regulate, andfine-tune humoral immune response- They are effector cells responsible for varioustypes of cell mediated immune responseslike; DTH, contact sensitivity, transplantationimmunity, and cytotoxicity
9 T Cell Surface Molecules TCR: A very diverse heterodimer that lacks a cytoplasmic tail that would allow direct cytoplasmic signaling once TCR binds an epitopeCD3 Complex: It is composed of a group of six invariant accessory molecules; one CD3 , one CD3 , two CD3ε, and an intracytoplasmic homodimer of ξ or CD247 chainsCytoplasmic signaling occurs through CD3 that noncovalently associate with TCR
11 The CD3 complex is essential for both cell surface expression of the TCR and for signal transduction once the TCR recognizes an antigenUnlike antibodies that can readily bind free antigen, a TCR cannot bind soluble antigens, but only enzymatically cleaved fragments of larger peptides presented as peptide MHC (pMHC) complexes
12 CD4 or CD8: Most mature T cells express CD4 or CD8 molecules, they function as important co-receptors in association with the TCRBy binding to invariant portions of the MHC I (CD8) or MHC II (CD4), they serve to increase the interaction of the MHC-bound antigenic fragment and the TCR
14 CD8 binds MHC class I CD4 binds MHC class II Most mature T cells are either CD4+ or CD8+. CD8 T cells kill cells infected with intracellular pathogens or tumor cellswhile CD4 T cells regulate (activate or suppress) other immune cells’ function.14
15 TCR Vs Immunoglobulin Both: Bind antigen Have Variable region and Constant regionHave a binding site that is a heterodimer (composed of 2 different chains)TCRs act only as receptorsIgs act as receptors and effector molecules(soluble antigen-binding molecules)
17 TCR StructureThe TCR is a member of the immunoglobulin supergene family and is composed of two polypeptide chains; a light α or chain and a heavy β or chainEach polypeptide chain of the heterodimer pair contains a variable and a constant region domainThe Vα and V regions are encoded by V and J gene clustersThe Vβ and V regions are encoded by V, D, and J gene clustersThe D gene cluster provides an additional source of variation
20 The gene clusters undergo DNA rearrangement, similar to that already described for immunoglobulin genes, to synthesize αβ dimers or dimersAs with immunoglobulins, the constant domain of the α and β or and chains are encode by constant region genes (Cα and Cβ or C or C )T cell receptors do not undergo any subsequent changes equivalent to isotype switch, and somatic hypermutation, important to generating diversity of immunoglobulins.
21 As might be imagined, in the random process of generating diversity, a variety of TCR specificities would be generated for peptides that one may never encounter during his lifetimeThree distinct categories of TCR specificities can be identified:- Those that recognize peptides that will never beencountered- Those that recognize peptides produced by potentialpathogens or peptides of foreign origin- Those that recognize peptides that are produced bycells of self
22 TcR a gene rearrangement by SOMATIC RECOMBINATION Germline TcR VnJCV2V1Rearranged TcR1° transcriptSpliced TcR mRNARearrangement very similar to the IgL chains
23 TcR a gene rearrangement RESCUE PATHWAY There is only a 1:3 chance of the join between the V and J region being in frameVnJCV2V1Vn+1chain tries for a second time to make a productivejoin using new V and J elementsProductivelyrearranged TcR1° transcript
24 TcR b gene rearrangement SOMATIC RECOMBINATION L & Vx52D1JC1D2C2Germline TcR D-J JoiningV-DJ joiningRearranged TcR 1° transcriptC-VDJ joiningSpliced TcR mRNA
25 TcR b gene rearrangement RESCUE PATHWAY There is a 1:3 chance of productive D-J rearrangement and a 1:3 chance of productive V D-J rearrangement(i.e only a 1:9 chance of a productive b chain rearrangement)D1JC1D2C2Germline TcR D-J JoiningV-DJ joiningV2nd chance atV-DJ joiningNeed to remove non productiverearrangementUse (DJC)b2 elements
26 TCR gene rearrangements occur in the thymus The same RSS and the same enzymes are used to rearrange both the TCR genes and the Ig genes. P and N nucleotides are added at the junctions between rearranged segmentsn=70-80n=52
27 TCR b Chain D region can be read in all frames GTACTGCAGATTVJDJ starts with ATT No additions: J is in frameGTACCTGCAGATTVJDOne addition and J is out of frame; the ATT start for J is lost (as are all appropriate downstream codons)GTACCTGCAGGATTVJDTwo addition and J is out of frame; the ATT start for J is lost (as are all appropriate downstream codons)GTACCTGCAGGCATTVJDThree addition and J is in frame; the ATT start for J is is present (as are all appropriate downstream codons)
28 -chain locus is first to be rearranged Wehfp wefnp ‘FFOEO
29 Two chances for productive (=correct reading frame) rearrangement: b chain
31 Multiple rounds of -chain rearrangement can rescue nonproductive TCR Wehfp wefnp ‘FFOEO
32 TCR gene rearrangement generates the TCR repertoire Pre-TCR complex stops further gene rearrangement at b locus, and induces thymocyte proliferationFinally TCR+ DP cells are made32
33 Germline configuration of g and d loci TCRd D, J and C exons are encoded in the intron between the the Vs and the Js of the TCRa locus. The V segments for TCRd (4 known) are mixed in with the V segments of the TCRa
35 Figure 3-8 part 2 of 2 Most gd T cells do not express CD4 or CD8. They are thought to be:First line of defense?Bridge between innate and adaptive responses?Figure 3-8 part 2 of 2
36 Signals through the TCR and the pre-TCR compete to determine thymocyte lineageWehfp wefnp ‘FFOEO36
37 Generation of diversity in the TcR COMBINATORIAL DIVERSITYMultiple germline segments In the human TcRVariable (V) segments: ~70, 52Diversity (D) segments: 0, 2Joining (J) segments: 61, 13The need to pair and chains to form a binding sitedoubles the potential for diversityJUNCTIONAL DIVERSITYAddition of non-template encoded (N) and palindromic (P)nucleotides at imprecise joints made between V-D-J elementsSOMATIC MUTATION IS NOT USED TOGENERATE DIVERSITY IN TcR
39 T Cell DevelopmentT cell precursors migrate from the bone marrow to enter the thymus as thymocytes, they express neither αβTCR nor CD4 or CD8 and are called double negative (DN) cellsDN cells proliferate in the subcapsular region of the thymus and differentiate to express low levels of newly generated αβTCR, both CD4 and CD8, and are called double positive (DP) cells
40 DP cells move inward to the deeper portion of the thymus, where they are fated to die within 3-4 days, unless their TCRs recognize an MHC class I or class II molecules on thymic dendritic cells. This process is called positive selectionAlthough the mechanism of positive selection is yet unclear, partial recognition of class II by CD4 or class I by CD8 molecules must occurT cells that recognize self MHC molecules survive
41 A DP thymocyte with a TCR that engage MHC class I may become a CD8+ T cell and a DP thymocyte that recognizes MHC class II may become a CD4+ T cellClass I and class II molecules are not displayed on cell surface unless they are loaded with a peptideOnly molecules of self origin are available on thymic APCs, and these are presented to the DP thymocyte in the deep or medullary area of the thymus
43 Thymocytes that show strong interaction with MHC molecules or pMHC complexes undergo apoptosis, a process known as negative selectionThymocytes that survive both positive and negative selection migrate from the thymus to populate lymphoid tissues and organs as T cells
45 Each thymocyte maturation stage occurs at a distinct location of the thymusWehfp wefnp ‘FFOEOYoung adult:~5x107 thymocytes produced/day~1.5x106 mature cells leave/day
46 Figure 5-3 part 1 of 2 DN (CD4-CD8-) and DP (CD4+CD8+) Immature thymocytes are hereDifferentiationMore mature SP (CD4+CD8-or CD8+CD4-) thymocytes are here
47 Positive SelectionPositive selection selects T cells that recognize peptides on self MHCThis is to assure that mature T cells can respond to antigen-presented on self MHC.Self MHC I and II harboring self peptides on thymic epithelial cells recognize and activate TCRs on some DP thymocytes.DP thymocytes should receive this signal within 3-4 days to survive, otherwise they undergo apoptosis.
48 Negative SelectionNegative selection eliminates T cells with TCRs that bind too strongly to self antigen/MHC complex (autoreactive cells are removed by this process)Dendritic cells and macrophages in cortico-medullary junction mediate it.Negative selection cannot eliminate T cells whose receptors are specific for self peptides that are present outside of the thymusThese cells enter circulation, but soon to be rendered anergic or unresponsive by other mechanims.
50 Does receptor occupancy explain positive and negative selection ? Low occupancySurvivalHigh occupancyNegative selection
51 Does the TIME of receptor occupancy explain positive and negative selection ?Long occupancyNegative selectionShort occupancySurvivalShort signaling Long signaling
52 Stage of maturation can be distinguished by the expression of specific surface moleculesDNDPWehfp wefnp ‘FFOEOSP
53 Types of T cells Conventional: Uses ab TCR Helper (CD4+) and cytotoxic (CD8+) T cellsMore abundant and highly specificRestricted by classical MHC (I and II) moleculesNon-conventional:Uses gd TCRPrimitive with broad specificityRestricted by non-classical molecules
54 CD4+ T cellsT cells with CD4 marker (glycoprotein) represent 70% of T cells in the peripheryNamed T helper cellsPlay central role in modulating cellular immunity via secretion of cytokines that mediate:B cell activationImmunoglobulin secretion (quality)Macrophage and dendritic cell activationCellular chemotaxis and inflammationTwo subsets; Th1 and Th2 cells
55 Th1 and Th2 cellsCD4+ T helper cells can be classified into two types based on their cytokine profiles: T helper cell type 1 (Th1) and T helper cell type 2 (Th2).Cytokine profile is influenced by several factors:Nature and dose of antigenRoute of administrationType of antigen presenting cell/ costimulationGenetic backgroundThe cytokine profile determines the effector function of the helper cell
56 Differentiation of naive CD4 T cells into different subclasses The differentiation of naive CD4 T cells into different subclasses of armed effector T cells is influenced by cytokines elicited by the pathogen. Many pathogens, especially intracellular bacteria and viruses, activate dendritic cells and NK cells to produce IL-12 and IFN-g, which cause proliferating CD4 T cells to differentiate into TH1 cells. IL-4 can inhibit these responses. IL-4, produced by an NK 1.1+ T cell in response to parasitic worms or other pathogens, acts on proliferating CD4 T cells to cause them to become TH2 cells. The mechanisms by which these cytokines induce the selective differentiation of CD4 T cells is now the subject of intensive study. They may act either when the CD4 T cell is first activated by an antigen-presenting cell or during the subsequent proliferative phase.
57 The nature and amount of ligand determine CD4 T cell functional phenotype The nature and amount of ligand presented to a CD4 T cell during primary stimulation can determine its functional phenotype. CD4 T cells presented with low levels of a ligand that binds the T-cell receptor poorly differentiate preferentially into TH2 cells making IL-4 and IL-5. Such T cells are most active in stimulating naive B cells to differentiate into plasma cells and make antibody. T cells presented with a high density of a ligand that binds the T- cell receptor strongly differentiate into TH1 cells that secrete IL-2, TNF-b, and IFN-g, and are most effective in activating macrophages.
59 Differences between Th1 and Th2 cells Produces type 1 cytokinesIL-2, IFN- , TNF-α, TNF-βActivates macrophages and DCs for intracellular killing of pathogensMediates CMIProduces type 2 cytokinesIL-4, IL-5, IL-10, IL-13Provides help to B cells in antibody responseMediates allergy and immunity to extracellular pathogens, including parasites
60 Cytotoxic T cellsT cells that express CD8 molecule on their surface and they represent 30% of T cells in the peripheryDestroy cells infected by intracellular pathogens and cancer cellsClass I MHC molecules (nucleated body cells) expose foreign proteinsTC cell releases perforin and granzymes, proteins that form pores in the target cell membrane; causing cell lysis and/or apoptosis
62 Effector molecules of T cell subsets The three main types of armed effector T cell produce distinct sets of effector molecules. CD8 T cells are predominantly killer T cells that recognize pathogen-derived peptides bound to MHC class I molecules. They release perforin (which creates holes in the target cell membrane), granzymes (which are proteases that act intracellularly to trigger apoptosis), and often the cytokine IFN-g. A membrane-bound effector molecule expressed on CD8 T cells is Fas ligand. When this binds to Fas on a target cell it activates apoptosis in the Fas-bearing cell. CD4 T cells recognize peptides bound to MHC class II molecules and are of two functional types: TH1 and TH2. TH1 cells are specialized to activate macrophages that are infected by or have ingested pathogens; they secrete IFN-g as well as other effector molecules, and express membrane-bound CD40 ligand and/or Fas ligand. These are both members of the TNF family but CD40 ligand triggers the activation of the target cell, whereas Fas ligand triggers the death of Fas-expressing cells, so their pattern of expression has a strong influence on their function. TH2 cells are specialized for B- cell activation; they secrete the B-cell growth factors IL-4 and IL-5. The principal membrane-bound effector molecule expressed by TH2 cells is CD40 ligand, which binds to CD40 on the B cell and induces B-cell proliferation.
63 Regulation of the Immune Response How does the immune system prevent self reactivity while maintaining reactivity to invaders/non-self?Clonal deletion/inactivation of auto-reactive cellsRegulatory T cells keep potentially pathogenic self reactive T cells in check through “suppressive” mechanisms
64 New T cell phenotypes Regulatory T cells Naturally occurring (CD25 positive)Induced (IL-10 and TGF-β)Some NK T cellsSuppressor T cellsTh17 cells: produce IL-17, role in acute inflammation, suppress Th2.
67 Regulatory T Cells (Treg) CD3+, TcR+, CD4+, CD25+FunctionSuppress the activity of effector Th and Tc cellsInhibition is antigen specific and MHC restrictedInhibition depends on cell:cell contactAppear to function in autoimmunityDevelopmentDevelop in the bone marrow from DP cellsArise as a result of strong binding to self MHC and self Ag during negative selection. Alternative to cell death or anergyDevelopment and maintenance are dependent upon B7
68 What are the requirements for Treg development? TCR engagement in the thymus (high affinity but not so high as negative selection)Higher percentage of thymocytes with high affinity TCR in context of auto antigen develop into regsT cells of appropriate affinity are “instructed” to become TregsSelective sparing of pre-committed cells from negative selection or promotion of Treg lineage development?higher percentage, but not higher absolute numberPreferential elimination of non-regulatory T cells rather than increased production of TregsTCR engagement serves as survival or expansion signal of Tregs pre-committed to that lineage.
69 The Treg cell phenotype CD4Co-receptor for TCR recognition of MHC II/AgCD25 IL-2RaIL-2R component, confers high affinity binding to IL-2RbgKey TR growth factorCTLA-4 cytotoxic T lymphocyte Ag-4Binds to B7s (CD80/86) on APC, acts as co-stimulatory molecule for TR (blocking CTLA-4 inhibits TR)GITR glucocorticoid induced TNF relatedproteinLigation inhibits TR function (agonist inhibit TR, blocking augments TR)FoxP3Forkhead /winged-helix TF critical for TR activity and developmentUnlike surface markers / receptors, TE do not express FoxP3CD4GITRCD25CTLA-4TregFoxp3The Treg cell phenotype
70 Modulation of immune responses by Treg cells Treg cells are crucial for the induction and maintenance of peripheral tolerance to self-antigensTreg cells can also suppress immune responses toTumor antigensAlloantigenAllergensMicrobial antigensTeffTregTumor ClearanceMicrobial ImmunityAutoimmunityTransplant rejectionGraft-versus host diseaseAllergyTeffTregSelf-toleranceTransplantation toleranceTumor ProgressionMicrobial PersistenceTeffTregSelf-toleranceImmunocompetenceSheng Cai