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

fere

nti

atio

n

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