A Mechanistic Investigation of T Cell Receptor- Mediated ...
T Cell Receptor & T Cell Development
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Transcript of T Cell Receptor & T Cell Development
T Cell Receptor &
T Cell Development
2
Questions for the next 2 lectures:
How do you generate a diverse T cell population with functional TCR rearrangements?
How do you generate a T cell population that is self-MHC restricted?
How do you ensure that those diverse T cell receptors are not-self reactive?
How do you coordinate lineage specification with MHC specificity and coreceptor expression? - vs. T cell - CD4 vs. CD8
T lymphocyte:a key regulator of the immune system
T Lymphocytes• Arise 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 nonself
• Thymocytes that cannot make this induction are eliminated, those that can may further differentiate, mature, and graduate as T lymphocytes and enter the circulation
Origin, generation and differentiation of T cells
T Cell Development• T 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
Figure 5-2Thymic 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.
T Cell Functions• Collectively, T cells display a number of diverse
functions:
- They often function to initiate, regulate, and
fine-tune humoral immune response
- They are effector cells responsible for various
types of cell mediated immune responses
like; DTH, contact sensitivity, transplantation
immunity, and cytotoxicity
T Cell Surface Molecules• TCR: A very diverse heterodimer that lacks a
cytoplasmic tail that would allow direct cytoplasmic signaling once TCR binds an epitope
• CD3 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 chains
• Cytoplasmic signaling occurs through CD3 that noncovalently associate with TCR
Figure 3-6TCR Complex
• The CD3 complex is essential for both cell surface expression of the TCR and for signal transduction once the TCR recognizes an antigen
• Unlike 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
• CD4 or CD8: Most mature T cells express CD4 or CD8 molecules, they function as important co-receptors in association with the TCR
• By 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
Figure 3-10The structures of CD4 and CD8
CD8 binds MHC class ICD4 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.
TCR Vs ImmunoglobulinBoth:
• Bind antigen
• Have Variable region and Constant region
• Have a binding site that is a heterodimer (composed of 2 different chains)
TCRs act only as receptors Igs act as receptors and effector molecules
(soluble antigen-binding molecules)
Similarity between TCR and Ig
TCR Structure• The 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 chain
• Each polypeptide chain of the heterodimer pair contains a variable and a constant region domain
• The Vα and V regions are encoded by V and J gene clusters
• The Vβ and V regions are encoded by V, D, and J gene clusters
• The D gene cluster provides an additional source of variation
Figure 3-7
and TCR gene loci (germline configuration)
• The gene clusters undergo DNA rearrangement, similar to that already described for immunoglobulin genes, to synthesize αβ dimers or dimers
• As 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.
• 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 lifetime
• Three distinct categories of TCR specificities can be identified:
- Those that recognize peptides that will never be
encountered
- Those that recognize peptides produced by potential
pathogens or peptides of foreign origin
- Those that recognize peptides that are produced by
cells of self
TcR gene rearrangement bySOMATIC RECOMBINATION
Spliced TcR mRNA
Germline TcR
Vn J CV2 V1
Rearranged TcR1° transcript
Rearrangement very similar to the IgL chains
TcR gene rearrangement RESCUE PATHWAY
There is only a 1:3 chance of the join between the V and J region being in frame
Vn J CV2 V1Vn+1
chain tries for a second time to make a productive join using new V and J elements
Productivelyrearranged TcR1° transcript
Rearranged TcR 1° transcript
Spliced TcR mRNA
L & Vx52 D1 J C1 D2 J C2
Germline TcR
TcR gene rearrangementSOMATIC RECOMBINATION
D-J Joining
V-DJ joining
C-VDJ joining
D1 J C1 D2 J C2
Germline TcR
D-J Joining
V-DJ joining
V
TcR gene rearrangement RESCUE PATHWAYThere 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 chain rearrangement)
2nd chance atV-DJ joining
Need to remove non productiverearrangement
Use (DJC)2 elements
TCR gene rearrangements occur in the thymus
n=70-80
n=52
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 segments
J starts with ATTNo additions: J is in frameGTACTGCAGATT
V JD
GTACCTGCAGATT
V JDOne addition and J is out of frame; the ATT start for J is lost (as are all appropriate downstream codons)
GTACCTGCAGGATT
V JD Two addition and J is out of frame; the ATT start for J is lost (as are all appropriate downstream codons)
GTACCTGCAGGCATT
V JDThree addition and J is in frame; the ATT start for J is is present (as are all appropriate downstream codons)
TCR Chain D region can be read in all frames
-chain locus is first to be rearranged
Two chances for productive (=correct reading frame) rearrangement: chain
-chain rearrangement
Multiple rounds of -chain rearrangement can rescue nonproductive TCR
TCR gene rearrangement generates the TCR repertoire
Pre-TCR complex stops further gene rearrangement at locus, and induces thymocyte proliferation
Finally TCR+ DP cells are made
Germline configuration of and loci
TCR D, J and C exons are encoded in the intron between the the Vs and the Js of the TCR locus. The V segments for TCR (4 known) are mixed in with the V segments of the TCR
Figure 3-8 part 2 of 2Most T cells do not express CD4 or CD8.
They are thought to be:First line of defense?Bridge between innate and adaptive responses?
Signals through the TCR and the pre-TCR compete to determine thymocyte lineage
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, 13
The need to pair and chains to form a binding sitedoubles the potential for diversity
JUNCTIONAL DIVERSITYAddition of non-template encoded (N) and palindromic (P) nucleotides at imprecise joints made between V-D-J elements
SOMATIC MUTATION IS NOT USED TO GENERATE DIVERSITY IN TcR
The Generation of Diversity (GOD)
T Cell Development
• T 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) cells
• DN 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
• 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 selection
• Although the mechanism of positive selection is yet unclear, partial recognition of class II by CD4 or class I by CD8 molecules must occur
• T cells that recognize self MHC molecules survive
• 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 cell
• Class I and class II molecules are not displayed on cell surface unless they are loaded with a peptide
• Only 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
CD4+CD8+ DP cells: To be CD4 or CD8?
• Thymocytes that show strong interaction with MHC molecules or pMHC complexes undergo apoptosis, a process known as negative selection
• Thymocytes that survive both positive and negative selection migrate from the thymus to populate lymphoid tissues and organs as T cells
Figure 5-3 part 2 of 2
Each thymocyte maturation stage occurs at a distinct location of the thymus
Young adult:~5x107 thymocytes produced/day~1.5x106 mature cells leave/day
Figure 5-3 part 1 of 2
Dif
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DN (CD4-CD8-) and DP (CD4+CD8+) Immature thymocytes are here
More mature SP (CD4+CD8-or CD8+CD4-) thymocytes are here
Positive Selection
• Positive selection selects T cells that recognize peptides on self MHC
• This 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.
Negative Selection• Negative 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 thymus
• These cells enter circulation, but soon to be rendered anergic or unresponsive by other mechanims.
Does receptor occupancy explainpositive and negative selection ?
High occupancyNegative selection
Low occupancySurvival
Does the TIME of receptor occupancy explainpositive and negative selection ?
Long occupancyNegative selection
Short occupancySurvival
Short signaling Long signaling
Stage of maturation can be distinguished by the expression of specific surface molecules
DN
DP
SP
Types of T cells• Conventional:
– Uses TCR– Helper (CD4+) and cytotoxic (CD8+) T cells– More abundant and highly specific– Restricted by classical MHC (I and II)
molecules
• Non-conventional: – Uses TCR– Primitive with broad specificity– Restricted by non-classical molecules
CD4+ T cells• T cells with CD4 marker (glycoprotein) represent 70%
of T cells in the periphery • Named T helper cells
• Play central role in modulating cellular immunity via secretion of cytokines that mediate:– B cell activation– Immunoglobulin secretion (quality)– Macrophage and dendritic cell activation– Cellular chemotaxis and inflammation
• Two subsets; Th1 and Th2 cells
Th1 and Th2 cells• CD4+ 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 antigen• Route of administration• Type of antigen presenting cell/ costimulation• Genetic background
– The cytokine profile determines the effector function of the helper cell
Differentiation of naive CD4 T cells into different subclasses
The nature and amount of ligand determine CD4 T cell functional phenotype
T Helper (CD4+ ) Subsets
Th2 response, Humoral Immunity and Acute Hypersensitivity
IL-4IL-10IL-13IL-5IL-6
Anti-Inflammatory
Cytokines
Th2
IL-4Th0
IFN-IL-2LT
Pro-InflammatoryCytokines
Th1
FasL
DR4
Th1 responseCellular Immunity
DTH
SuicideFas
AntigenAPC
IL-12
Differences between Th1 and Th2 cells
• Produces type 1 cytokines– IL-2, IFN- , TNF-α,
TNF-β• Activates macrophages
and DCs for intracellular killing of pathogens
• Mediates CMI
• Produces type 2 cytokines– IL-4, IL-5, IL-10, IL-13
• Provides help to B cells in antibody response
• Mediates allergy and immunity to extracellular pathogens, including parasites
Th1 cell Th2 cell
Cytotoxic T cells• T cells that express CD8 molecule on their surface
and they represent 30% of T cells in the periphery
• Destroy cells infected by intracellular pathogens and cancer cells
• Class I MHC molecules (nucleated body cells) expose foreign proteins
• TC cell releases perforin and granzymes, proteins that form pores in the target cell membrane; causing cell lysis and/or apoptosis
Effector molecules of T cell subsets
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 cells
–Regulatory T cells keep potentially pathogenic self reactive T cells in check through “suppressive” mechanisms
New T cell phenotypes
• Regulatory T cells– Naturally occurring (CD25 positive)– Induced (IL-10 and TGF-β) – Some NK T cells– Suppressor T cells
• Th17 cells: produce IL-17, role in acute inflammation, suppress Th2.
Bettelli et al., Nature 2008
Th17 and TregTh17 and Treg
Regulatory T Cells (Treg)• CD3+, TcR+, CD4+, CD25+
• Function– Suppress the activity of effector Th and Tc cells– Inhibition is antigen specific and MHC restricted– Inhibition depends on cell:cell contact– Appear to function in autoimmunity
• Development– Develop in the bone marrow from DP cells– Arise as a result of strong binding to self MHC and
self Ag during negative selection. Alternative to cell death or anergy
– Development and maintenance are dependent upon B7
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 regs – T cells of appropriate affinity are “instructed” to
become Tregs• Selective sparing of pre-committed cells from negative
selection or promotion of Treg lineage development? – higher percentage, but not higher absolute number– Preferential elimination of non-regulatory T cells
rather than increased production of Tregs– TCR engagement serves as survival or expansion
signal of Tregs pre-committed to that lineage.
Treg
CD4 CD25CTLA-4
GITR
The Treg cell phenotype
• CD4– Co-receptor for TCR recognition of
MHC II/Ag• CD25 IL-2R
– IL-2R component, confers high affinity binding to IL-2R
– Key TR growth factor• CTLA-4 cytotoxic T lymphocyte Ag-4
– Binds to B7s (CD80/86) on APC, acts as co-stimulatory molecule for TR (blocking CTLA-4 inhibits TR)
• GITR glucocorticoid induced TNF related protein
– Ligation inhibits TR function (agonist inhibit TR, blocking augments TR)
• FoxP3– Forkhead /winged-helix TF critical for TR
activity and development– Unlike surface markers / receptors, TE
do not express FoxP3
Foxp3
Modulation of immune responses by Treg cells
• Treg cells are crucial for the induction and maintenance of peripheral tolerance to self-antigens
• Treg cells can also suppress immune responses to
1. Tumor antigens
2. Alloantigen
3. Allergens
4. Microbial antigens
Teff
Treg
Tumor ClearanceMicrobial Immunity
AutoimmunityTransplant rejection
Graft-versus host diseaseAllergy
Teff
Treg
Self-toleranceTransplantation tolerance
Tumor Progression Microbial Persistence TeffTreg
Self-toleranceImmunocompetence
Sheng Cai