Chapter 7 - Development of T Lymphocytes

Chapter 7

• The development of T cells in the ________

- Stages of gene rearrangement that produce the 1º repertoire of T-cell receptors

• Positive and negative selection of T-cell repertoire

- Processes of + & - selection that act on the primary repertoire of T-cell receptors in the thymus to produce the circulating population of ________; naïve T cells

thymus

mature

The Development Of T Cells Compared To The Development Of B Cells

• Similarities in the development of T and B lymphocytes:

• Derive from _______ marrow stem cells

• Undergo gene __________ -> antigen receptors

• B cells rearrange in the bone _________

• Precursors of T cells leave the bone marrow ->__________

• The formation of 2 distinct T cell lineages

• _____ Receptors (1-2% of the primary repertoire leave the thymus) (B rearranges first)

• ______ Receptors (not as stringent selection, don't need mhc, no positive selection)

bone

rearrangement

marrow

thymus

α:β

δ:γ

Primary Lymphoid Tissue For T Cell Development Is The Thymus

• Major function of the thymus function is to ensure that ________ T cells that leave thymus are restricted to the particular MHC class expressed by an individual person (self -MHC)

• Two selection processes:

• Positive selection leads to the death of immature T cells having receptors that do not interact with any self-______ class I and II (must moderately interact w MHCs)

• Negative selection induces the death of those immature T cells that are ___________ (receptors bind too strongly to a self-MHC molecule)

• Mature T cell leaving the thymus to circulate in the secondary lymphoid organs is:

1. Rendered ___________ of self-antigens

2. Responsive to ________ antigens

3. Ready to _________ infection

mature

MHC

autoreactive

tolerant

foreign

fight

T cell rearranges its T cell receptor genes in the _________

Immature T cells that recognize self MHC receive signals for survival. Those that interact strongly with the self antigen are ___________ from the repertoire

Mature T cells encounter foreign antigens in the peripheral lymphoid organs and are ________

Activated T cells proliferate and migrate into _________ sites to eliminate infection

Only __-___% of α:β T cell primary repertoire exit the thymus and go into circulation

thymus

removed

activated

peripheral

1-2

The Development Of T Cells In The Thymus

• T cells originate from _______ _________ as stem cells

• Emigrate to mature in the ________

• Named thymus-dependent lymphocytes -> T cells

• Majority are _____ cells (B first, delta found on alpha chain between VJ)

• Minority are _____ cells

• 2 lineages develop in _________ from common precursors

While developing in the thymus

• T cells also express other cell-surface proteins related to their eventual functions

• Examples are _______ and _______ glycoproteins

bone marrow

thymus

α:β

δ:γ

parallel

CD4, CD8

T Cells Derive From Bone Marrow Stem Cells

• From the bone marrow, T cell precursors migrate through the ______ to the _______

• Thymus is where the __________ of T cells occurs

• Not ________

• No ________ connection

• Mature T cells then leave the thymus in the blood and enter these ___________ lymphoid organs, such as the spleen or lymph nodes.

• In the absence of activation mature T cells recirculate between the ___________, the __________ lymphoid tissues, the lymph and the ________

blood, thymus

development

activation

lymphatic

secondary

blood, secondary, GALT

T Cells Develop In The Thymus

• Thymus is found in the upper anterior thorax above the ________

• Immature T cells - called _________ - are embedded in an epithelial cell network called the ________ _______

• Thymus is primary lymphoid organ:

- Involved in the ___________ of T cells

- Not involved in lymphocyte __________ via lymph

- ________ is the only route through which T cells enter and exit

heart

thymocytes, thymic stroma

development

recirculation

Blood

T Cells Develop In The Thymus

• In the embryonic development of thymus

- Epithelial cells of cortex  _______

- Epithelial cells of medulla  _______

- Rudimentary thymus called thymic _______

• Is colonized by cells from bone marrow

• Progenitor cells -> thymocytes & dendritic cells -> populate ________

• Bone marrow derived macrophages  also populate __________ (also macrophages scattered throughout the _______ of thymus)

• Thymocyte mature -> progressively move from the outer subcapsular region to the ________ cortex and the medulla

outer

inner

anlage

medulla

medulla, cortex

inner

The Epithelial Cells Of The Thymus Form A Network Surrounding Developing Thymocytes

• Scanning electron micrograph of thymus

• Developing thymocytes(spherical cells) occupy the interstices of an extensive network of __________ cells

epithelial

2 thymus stromal cells

cTEC - cortical thymic epithelial cells

mTEC - medullary thymic epithelial cells

cortex/cTEC:

medulla/mTEC:

matures from N -> S

somatic recomb B and a

pos selection

neg selection

The Cellular Organization Of The Thymus

• ____________ in both cortex and medulla remove the many thymocytes that fail to mature properly

Hassall’s corpuscles

• Characteristic feature of The _________

• (? Sites of cell __________?)

 Thymus= multi-lobal (stained with __________ and _________, viewed by light microscope)

 The darker staining of _________ vs with the lighter stained _________

Macrophages

medulla

destruction

hematoxylin, eosin

cortex, medulla

DiGeorge's Syndrome

• Example of importance for thedevelopment a functional T-cellrepertoire

• A deletion in chromosome ____ in which the __________ fails to develop and T cells are absent

• Susceptibility to wide range of opportunistic infections -> resembles _______ (severe combined immunodeficiency disease)

- affects both innate and adaptive

- cannot fix w bone marrow transplant

22, thymus

SCID

Thymus And Aging

• Thymus fully ____________ before birth; is most active in the _________; atrophies with ______

• Progressively shrinks, fat gradually claiming areas once packed with thymocytes = ___________ of the thymus

• Reduced production of new T cells due to aging does not noticeably __________ T cell immunity

• Nor does _________ = removal of the thymus affect T cell immunity of adults

they self renew

developed, young, age

involution

impair

thymectomy

Thymus And Aging

• Once established, the repertoire of mature peripheral T cells is _______ lived and/or _______-renewing

• Differs from the mature B-cell =_______ lived cells that are continually being replenished from the _______ marrow

long, self

short, bone

The Two Lineages Of T Cells Arise From A Common Thymocyte Progenitor

• Maturation of thymocytes into mature T cells occurs in distinct steps

• Marked by changes in the _______ of the TCR genes

• Expression of the _______ protein

• Production of other T-cell surface glycoproteins _____, ______, _______ complex

• Changes in cell surface __________ expressed at each developmental stage is a way to distinguish between subpopulations of developing thymocytes

status

TCR

CD4, CD8, CD3

proteins

double neg

double pos

no CD4 or CD8

both CD4 and CD8

The Two Lineages Of T Cells Arise From A Common Thymocyte Progenitor

• Progenitor T cells that enter the thymus lack the cell surface glycoproteins (CD4, CD8, CD3) of mature T cells, but they do have _______ (a cell surface glycoprotein of stem cells).

• The TCR genes are in germline configuration

• Upon interaction with thymic stromal cells, the progenitor T cells will proliferate and ___________

• Approximately one week later, progenitor T cells will express the T-cell specific adhesion molecule _______ and other surface markers such as ________ but no TCR complex

• No CD4 or CD8 -> called “double __________” thymocytes

• IL-7 receptor on T-cells is essential for binding ______ secreted by thymic stromal cells – helps tell the T-cell what to do next in its maturation.

CD34

differentiate

CD2, CD5

negative

IL-7

• __________ – at all stages of maturation in the thymus signals are sent through this receptor to drive theT-cell in their differentiation.

• Keeps the T cell on the T cell _______ not allowing the _______ genes to be opened up for rearrangement

Notch 1

path, BCR

uncommited progenitors

commited adhesion and signaling

TCR co receptors (double neg)

CD34 + CD44

CD2 + CD5

CD4 + CD8

notch ligand found on _______

________ binds to ligand then gets cleaved and moves into intracellular domain of thymocyte ( to keep on T cell path)

cTEC

Notch 1

T Cells Have Two Lineages Distinguished By TheExpression Of An α:β Or A γ:δ TCR

• Commitment does not occur before TCR rearrangement but is a _______ to obtain a productive rearrangement

• Thymocytes will rearrange their ____, ___, ____ chain genes at about the same time

• Different from B-cell development (recall: each type of Ig gene is rearranged in turn and in a set _______)

• Productive γ and δ-chain gene rearrangement made prior to a productive β-chain rearrangement leads to ____ receptor which signals cell to stop rearrangement of ___ chain

• More frequently the ____ chain productively rearranges before the γ and δ-chains

• β-Chain assembles with a surrogate ___ chain = ptα (pre-T-cell receptor) which signals the cell to halt rearrangement of ____, ____, ____-chain genes and begin toproliferate

race

β, γ, δ

order

γδ, β

β

α

β, γ, δ

beta gets ____ rearrangements

gamma gets ____ rearrangements

delta is ______ out if alpha rearranges properly

2

4

cut

Pre-TCR 4 signals

(once has a mature β chain)

- stops additional TCR ____-chain locus rearrangement

- stimulates __________

- stimulates _____ and ______ expression

- cell becomes permissive for TCR ____-chain locus rearrangements

β

proliferation

CD4, CD8 (double pos)

α

T cells have two lineages distinguished by the expression of an αβ or a γδ TCR

• After expression of the pre-TCR (mature heavy chain), the recombination machinery is reactivated & and targeted towards the ___ chain loci (and the γ and δ loci)

• In a ________ of these cells, successful completion of γ and δ chain gene rearrangements occurs before the α chain gene has rearranged -> the γδ lineage

• In a _________ of these cells, productive rearrangement of the α-chain gene occurs first -> an αβ T cell

• The δ chain locus is located within the ___-chain locus...a rearrangement at an α-chain locus results in the deletion of the complete ____ chain locus from the chromosome

α

minority

majority

α, δ

T Cells Have Two Lineages Distinguished By The Expression Of An αβ Or A γδ TCR

Cells committed to one lineage can contain productive rearrangements forthe TCR genes of the _______ lineage

(except for ___-chain)

pre TCR: double neg -> double pos

pos selection: double pos -> single pos

α

Immature T Cells That Undergo Apoptosis Are Ingested By Macrophages In The Thymic Cortex

• Failure to make a productive rearrangement results in death by apoptosis (fate of about ____% of thymocytes)

• __________ in thymus continually remove dead cells

• Cells have been stained for apoptosis with a _____ dye

• Apoptotic cells are throughout the _______ but are rare in the _________

• Higher magnification _____ for apoptotic cells and ______ for macrophages

• __________ cells are visible within macrophages

98%

Macrophages

red

cortex, medulla

red, blue

Apoptotic

Production of T-cell receptor β chain stops β-chain rearrangement and leads to expression of CD4 and CD8

• _____ Receptor most abundant type found on T cells.

• TCR β-chain locus = variable(____), diversity (____), and joining (____) gene segments and is rearranged first (similar to heavy chain in Ig’s)

• TCR α-chain locus has no _____ segments and is rearranged after the β-chain (similar to light chain in Ig’s)

αβ

V, D, J

D

Production of T-cell receptor β chain stops β-chain rearrangement and leads to expression of CD4 and CD8

• Production of a functional β-chain gene -> β-chain translation and assembly with a __________ α-chain (preTα), _______ proteins and ____ chain to form a pre-T cell receptor -> transported to the cell surface

• Role of pre –T-cell receptor is analogous to the pre-B-cell receptor in ____-cell development

• Triggers the thymocyte to __________ and halt ___-chain gene rearrangement

• Ensures only ________ type of T-cell receptor β-chain is expressed by the T cell

surrogate, CD3, ζ

B

proliferate, β

one

Unproductive Rearrangement At One β-Chain Locus Can Lead To Rearrangement Of The β-Chain On the Homologous Chromosome, Rearrangement At the Same Locus Can Also Occur

• This is not the case for Ig ____-chain genes

• This chance exists with T-cell β-chain because 2 sets of _______ and _____ gene segments are tandemly associated with the Vβ gene segments.

• Potential to “try out” up to ___ gene rearrangements per cell = _____% of T cells make successful rearrangement of the β-chain gene.

• Only _____% success rate for productive H-chain gene rearrangement by B cells.

* image on slide 29

H

DβJβ, Cβ

4, 80%

55%

B chain gets up to 2 rearrangements

___ max arrangements par cell ( 2 upstream 2 downstream)

4

Unproductive Rearrangement At One β-Chain Locus Can Lead To Rearrangement Of The β-Chain On the Homologous Chromosome, Rearrangement At the Same Locus Can Also Occur

• Successful rearrangement of a β-chain gene induces expression of the two co-receptors (______ and ______) (double neg to double pos)

• Called “double-__________” thymocytes

• Found predominantly in the _______ cortex of the thymus

• Interact intimately with the network of ___________ cells

• During cell proliferation initiated by signaling through the pre-TCR, expression of the _______ and _________ genes is repressed (allelic exclusion of the beta chain)

• No rearrangement of the α-chain genes occurs until the double-positive cells stop ________

• Ensures each cell with a productive β-chain gene rearrangement produces many daughter cells that have the potential to express a different ____-chain gene

CD4 & CD8

positive

inner

epithelial

RAG-1, RAG-2

dividing

α

T-cell Receptor α-Chain Genes Can Undergo Several Successive Rearrangements

• The TCR α-chain can undergo ___________ successive gene rearrangements

• The presence of many Vα and over 60 Jα gene segments allows many successive rearrangements (like Ig ____ in B cells)

• Almost every developing T cell will make a productive ___-chain rearrangement

several

κ

α

Checkpoints in T-cell Development

• Check-point 1 - Once a ________ chain is produced it is sent to the ER to make sure it can bind to the surrogate alpha chain (pTα) – __________

• Check-point 2 – Once an _______ chain is made it is sent to the ER to make sure it can bind to the beta chain- _______

beta, Pre-TCR

alpha, TCR

CD4 and CD8 (_______ of 2 stay on)

RAG 1, RAG 2, TdT ( ____ and _____)

ZAP70 and CD3 (always ____!)

one

on, off

on

Positive And Negative Selection Of The T-cell Repertoire

• The second phase of T cell development involves the selection of T cells bearing TCRs that can recognize an individual’s own _______ presenting peptides.

• The T-cell receptors and the MHC molecules have been interacting for millions of years and evolving together.

• Over that time the ___ gene segments (which code for CDR 1 and 2) have evolved to recognize the thousands of different MHC molecules across the human population (HLA’s).

• Remember that CDR 1 and 2 recognize the _______ molecule while CDR 3 recognizes the ___________.

• The V gene segment chosen may _______ produce CDR 1 and 2 that recognize the specific MHC of that person.

• This (pos) selection process involves only ______ T cells and not γδ (bc don't need MHC) T cells

MHC

V

MHC, peptide

not

αβ

T-cells That Can Recognize self-MHC Are Positively Selected In The _______ of the Thymus

• Gene _____________ produces a repertoire of T cells bearing TCRs that can interact with the thousands of MHC class I and II molecules present in a population.

• Therefore, TCRs expressed on the T cells of one individual are not made to specifically interact with _______ that individual's MHC molecules.

• Only a small population (2%) of double ___________ thymocytes will be able to bind to a specific individuals MHC’s and the rest die by apoptosis in the cortex.

• Positive selection is the process by which this small population of T cells that reacts with the individual’s own ________ molecules is selected

Cortex

rearrangement

only

positive

MHC

T-cells That Can Recognize self-MHC Are Positively Selected In The Thymus

• Positive selection takes place in the cortex of the thymus and is mediated by __________ epithelial cells bearing complexes of class I and class II self-MHC and self-peptides.

• Cortical epithelial cells form web of cell processes that envelope _______, _______ double-positive thymocytes.

• At the point of contact – interactions between the ________ of thymocytes with self-_______ and self-________ are tested

 If a peptide:MHC complex is bound by a thymocyte within ___-___ days of expressing a functional TCR, then a positive signal is delivered to the thymocyte

 A thymocyte that does not receive a signal dies by ___________ and is removed by macrophages.

- during those 3-4 days it leaves RAG1 and RAG2 on to make a second a chain to ensure positive interaction

(technically breaks allelic exclusion, functionally we don't)

cortical

CD4, CD8

αβTCR, MHC, peptide

3-4

apoptosis

T-cells that can recognize self-MHC are positively selected in the _______ of thymus

cortex

T-cells that can recognize self-MHC are positively selected in the thymus

• Self-peptides presented in the MHC molecules of cortical epithelial cells are derived from self-proteins present in the thymus.

• The number of different peptides that can be presented by oneindividual’s MHC molecule is estimated to be about 10,000.

• For someone who is heterozygous for the six major HLA genes about 180,000 self-peptides could be presented by ____ different MHC class I and II molecules.

• The mature T-cell repertoire is tens of millions or more, so most of the self-peptides:MHC complex will bind a T cell receptor during positive selection.

18

pos selection in cortical epithelial cells in the thymus looks for _________ to _________ binding

if weak - apoptosis

pos selection looks for TCR recognition of MHC and is when double pos becomes single pos

moderate, strong

double ______ -> double ______ (Pre TCR/ CD4+CD8) -> _______ pos ( CD4 or CD8)

neg, pos, single

Positive Selection Controls Expression Of The CD4 Or CD8 Co-receptor

•Positive selection also determines whether a double positive thymocyte matures into a CD8 or CD4 T cell, known as “__________-positive” thymocytes.

•CD4 T cells only interact with MHC class ___ molecules and CD8 T cells interact with MHC class ___ molecules.

•During positive selection...

-When a CD4 CD8 double-positive thymocyte interacts through its TCR with a class I MHC molecule, _______ is recruited and CD4 is excluded.

-When a CD4 CD8 double-positive thymocyte interacts through its TCR with a class II MHC molecule, _______ is recruited and CD8 is excluded.

single

II, I

CD8

CD4

Rearrangement Of α-Chain Stops Genes Once A Cell Has Been Positively Selected

• Rearrangement of the ____-chain locus continues throughout the 3-4days of positive selection – hence a T cell can ________ the specificity of the TCR it expresses.

• Once a T cell is positively selected, rearrangement of the α-chain ______.

• Some double positive thymocytes can express _______ α-chains (one frommaternal allele and one from the paternal allele) and thus two types of TCR and undergo ___________ selection by engagement of one of these receptors.

• The number of positively selected cells is very small; therefore, it is __________ that one cell will have two selected TCRs (one receptor is usuallynonfunctional).

Can you have 2 dif TCR on the T cell during pos selection?

α, change

stops

two, positive

rare

YES!

T-cells Specific For Self-antigens Are Removed In The Thymus By Negative Selection

• Negative selection serves to delete T cells whose antigen receptors bind too ___________ to the complexes of self-peptides and self-MHC molecules presented by thymic cells.

• ___________ Tolerance – Tolerance to self gained in the primary lymphoid tissue

• Negative selection is mediated by several cell types, most important ofwhich are the bone marrow-derived ___________ cells and ___________.

• Engagement of the MHC molecule of one of these specialized thymic antigen-presenting cells by the TCR of a thymocyte causes that cell to undergo apoptosis and phagocytosis by ______________.

neg selection occurs in the medulla (mTEC)

strongly

Central

dendritic, macrophages

macrophages

T-cells Specific For Self-antigens Are Removed In The Thymus By Negative Selection

• Negative selection cannot eliminate T cells bearing TCRs that can bind to self-peptides not present in the _________ (only does neg selection for TCRs in the thymus)

• _________ autoimmune regulator – used by medullary thymic epithelial cells to create self-peptides that are normally only found in the __________.

• Such cells enter the periphery but are rendered ____________ or inactivated.

• How the positive and negative selection processes used in the thymus lead to death or growth is not well understood but likely involves differences in the ___________ of the ligand-receptor interactions.

• Because of the diversity of HLA types in a population, an individual’s Tcell component becomes highly _________

thymus

AIRE, periphery

anergic

affinity

personalized

Regulatory T cells

CD4 T-cells Express

• _______

• ________, a transcriptional repressor, is used by the regulatory T-cells (unique to regulatory T-cells)

• Distinct from naïve T-cells

• Contact with MHC II – self-antigen can suppress ___________ of naïveT-cells responding to self-antigens

• Suppressive effects require contact between the ______ T-cells and secretion of non-inflammatory cytokines ________ and ________

CD25

FoxP3

proliferation

two, IL-10, TGF-B

T-cells Undergo Further Differentiation In Secondary Lymphoid Tissues After Encounter With Antigen

• The T cells that survive the selection processes in the thymus become _________, naïve T cells that recirculate through blood into the ___________ lymphoid organs.

• Mature T cells are _________-lived than B cells and in the absence of specific antigen stimulation will continue to circulate in the body for many years.

• The T cell zones of lymphoid organs are sites where naïve T cells are activated by __________ – which provokes the final phase of T-cell development and differentiation.

• Mature T cells become effector cells that can stay in __________ tissues or migrate to sites of ________.

mature, secondary

longer

antigen

lymphoid, infection

T-cells undergo further differentiation in secondary lymphoid tissues after encounter with antigen

• There are several different types of _________ T cells.

• CD8 T cells become activated _________ T cells.

• CD4 T cells differentiate under the influence of cytokines into _______ or _______ helper T cells.

• Which type of CD4 T cell predominates depends on the nature of the pathogen and the immune response needed.

• In a healthy individual there are about __________ the number of CD4 T cells to CD8 T cells.

• In patients with AIDS this proportion changes because the AIDS virus infectsand kills _______ T cells where they have less than 200

effector

cytotoxic

TH1, TH2

twice

CD4