Lecture 10 - Development of T lymphocytes

Lymphopoiesis

production of new lymphocytes in central lymphoid organs

why focus on T and B cell development?

1. only cells to rearrange their DNA to create unique receptors

2. they define specific immunity

3. clinical relevance

once an antigen receptor has formed, the cell undergoes _______ ______ to select for useful antigen receptors

rigorous testing 

what rigorous testing does the cell undergo once an antigen receptor has formed

SPECIFICITY and AFFINITY for self ligands and self MHCwhat

what is the primary site of T-Cell Development

Thymus

steps of development

common lymphoid progenitors (in bone marrow) → immature T-cell precursors (blood circulation) → Thymocytes (Thymus) 

what happens to thymocytes in the thymus

• develop specific T cell markers (TCR, CD3, CD4, or CD8) 

• thymic selection/education

two important parts of the thymus

medulla and cortex

outer layer of thymus

cortex

middle layer of thymus

medulla

what is present in the cortex

immature thymocytes + scattered macrophages

what is present in the medulla

More mature thymocytes + macrophages + DCs + few B cells

what makes up the Thymic Stroma

Cortical epithelial cell

Medullary epithelial cell

T-cell precursors proliferate extensively in the ______, but most _____ _____

T-cell precursors proliferate extensively in the thymus, but most die there

what percent of thymocytes die by apoptosis and what percent leave the thymus each day

98% die by apoptosis

2-4% leave the thymus each day

key players in apoptosis of thymocytes

macrophages in the thymic cortex and medulla

why does the large loss of thymocytes occur

because each thymocyte is carefully tested to make sure it can recognize self-MHC with self-peptides and that it won’t react strongly against the body’s own tissues - self-tolerance

Distinct ______ of thymocytes are produced in the thymus

Lineages

successive stages in thymocyte development are marked by

a. status of TCR genes

b. changes in TCR expression 

c. changes in cell-surface molecule expression (e.g. CD3, CD4, CD8)

particular combinations of cell-surface proteins are used as ________ for T cells at _________________

particular combinations of cell-surface proteins are used as markers for T cells at different stages of differentiation

two distinct lineages:

1. 𝛾δT cells

2. αβ T cells - develop into CD4 and CD8 T cells

what does double negative mean

early stages of Thymocyte development

• these cells lack both CD4 and CD8

• CD3^-CD4^-CD8^-

where are double negative T cells found

in the subscapular zone of cortex

what are the 4 DN stages

• ETP (DN1)

• DN2

• DN3

• DN4

what is ETP

early thymic progenitor

what is DN2

stage where cells become irreversibly committed to the T-cell lineage

what is DN3

TCR β-chain rearrangement; a small percentage become γδ T cells

what percentage of cells become γδ T cells

~5%

what stage do cells determine if they become γδ T cells

after DN2 and before DN3

what is DN4

successful β-chain expression (pre-TCR)

what happens after DN4

double positive stage

what is DP

double positive

• these cells express both CD4 and CD8

• CD3⁺CD4⁺CD8+

what cells progress from DN3→ DN4 → DP →

αβ T cells

what happens at the DP stage

positive and negative selection happen here

what comes after DP stage

single positive (SP) stage

what does SP mean

cells express either CD4⁺ or CD8⁺

after selection, what percentage of cells survive

less than 5% survive

what do the cells that survive become

CD4⁺ SP T cells (helper T cells)

CD8⁺ SP T cells (cytotoxic T cells)

what are helper T cells

CD4⁺ T cells

what are cytotoxic T cells

CD8⁺ T cells

where are double positive cells found

in the inner cortex

where are single positive cells found

in the medulla

when does commitment to T-cell lineage occur 

occurs in thymus after Notch signaling

in the thymus, what are CLP (common lymphoid progenitors) know as

ETPs (early thymic progenitors) a.k.a DN1

what do ETPs receive Notch signalling from

ETPs receive Notch signaling from thymic epithelial cells (TECs)

what is Notch signaling essential for

• essential for T cell lineage commitment

when is Notch signaling required for

required during early phases of thymocyte differentiation up to DN3 stage 

what happens to Notch signaling in DN4

during B-selection (DN4), Notch signaling turned off as a consequence of pre-TCR signaling

Features of Double-Negatives

• have NOT yet rearranged TCR loci

• do NOT express CD4 or CD8 

• Are the earliest T-Cell precursor in the Thymus

Markers:

• c-KIT - receptor for stem cell growth factor 

• CD44 - adhesion molecule - homing to thymus

• CD25 - alpha chain of IL2 receptor 

when does CD3 expression first appear

between DN2 and DN3

a small fraction of DN2 matures into _________, while most cells proceed to become _______

a small fraction of DN2 matures into γδ T cells, while most cells proceed to become ab TCR

DN2 begins to rearrange _________

B-chain locus

In DN3, newly formed B chain combines with ______ and _______to form the _______

In DN3, newly formed B chain combines with Pre-Ta (surrogate chain) and CD3 to form the Pre-T cell receptor

if B chain arrangement productive →

proceed to DN4; CD4 and CD8 expression is initiated

what is initiated at DN4

CD4 and CD8 expression is initiated

DP begins to rearrange what

alpha chain locus → low levels of aBTCR and CD3 → ready for selection

what does Notch signaling signal

become a T cell

when is Notch signaling expressed

early in DN1-DN3

what kind of signal is Notch signal

commitment signal: directs progenitor entering thymus to become T cells instead o B or NK cells

what happens without Notch signaling

thymocytes fail to adopt the T-cell lineage

CD3 signaling

signal through your TCR

when does CD3 signaling appear

at DN→ DP transition

what is CD3 apart of

part of the TCR complex, responsible for signal transduction once the TCR recognizes peptide-MHC

what does CD3 indicate

indicates that the pre-TCR or TCR complex is forming and functional

IL-7R (interleukin-7 receptor) signal

“stay alive and divide”

when is IL-7R signal expressed

mainly in early DN (DN1-DN3)

what does IL-7R signal provide

provides survival and proliferation signals during early thymocyte growth

when is IL-7R downregulated

downregulated in DP, since IL-7 signaling is no longer needed once TCR selection begins

RAG-1/2 signal

“build your TCR”

when is RAG1/2 active

during DN2-DN3 and again at DP

what does RAG1/2 encode

encodes enzymes that cut and join V(D)J segments during TCR gene rearrangement 

when is RAG1/2 turned off

after successful rearrangement to prevent further DNA breaks

why is Pre-TCR important

• indicate successful (productive) TCR B-chain rearrangement

• drives proliferation and maturation of thymocytes

• signals without a ligand (constitutive signaling)

• stops further B chain rearrangement - maintains allelic exclusion 

• triggers TCR a-chain rearrangement 

• promotes transition to the CD4⁺CD8⁺ DP stage

once a productive rearrangement at the B-chain locus has taken place, events occur to prevent what

further recombination of the B chain

what is this recombination shutdown referred to as

allelic exclusion

why is allelic exclusion vital

bc it prevents the thymocyte from producing more than one functional B chain as it continues development

Key message: Pre-TCR (pre-T-cell receptor) =

B-chain checkpoint that drives proliferation and triggers a-chain rearrangement

After B chain rearrangement is complete, ____ cells progress to _____

DN3 cells progress to DN4

what is expressed after B chain rearrangement

both CD4 and CD8 are expressed = now cells are DP (double positive)

expression of CD4 and CD8 initiates what

the rearrangement of a-chain locus in DP cells

what does a-chain pair with and what does this form

a-chain pairs with the already functional B-chain, forming a complete aBTCR on the cell surface

at DP stage, ____ and associated ____ complex are expressed at low levels

TCR and associated CD3 complex are expressed at low levels,

TCR and associated CD3 complex are expressed at low levels during DP stage, what is this just enough for?

just enough for the cell to test whether that receptor can recognize self-MHC molecules presenting self-peptide

what does this testing of whether the receptors can recognize self-MHC molecules mark

this marks the start of the selection process

what happens during the selection process

thymocytes that can moderately recognize self-MHC receive survival signals (positive selection), while those that can’t or react too strongly will die (negative selection)

Key message: DP thymocytes rearrange:

rearrange the a-chain, form a low-level aBTCR-CD3 complex, and enter selection to test MHC recognition

during which stage of thymocyte development does signaling through Pre-TCR first occur, and what is its main function?

a) DN2; initiates B-chain rearrangement 

b) DN3; tests for a functional B-chain and triggers proliferation 

c) DN4; begins a-chain rearrangement and allelic exclusion 

d) Double-positive (DP); promotes expression of CD4 and CD8

b) DN3; tests for a functional B-chain and triggers proliferation

what developmental stage is mostly found in the subscapular region of the outer cortex

mostly DNs

what stage cells are mostly found in the deeper cortex

mostly DPs

what stage cells are mostly found in the medulla

mostly SPs

what do cTECs (cortical epithelial cells) express

MHC and MHC II - role in POSITIVE SELECTION

what do mTECs (medullary epithelial cells) do

present peripheral antigens - role in NEGATIVE SELECTION

how many attempts can be made to produce a functional B-chain

up to 4 attempts can be made to produce a functional B-chain

both TCR a and B loci are in ______________

germline (unrearranged) form

• cell: early DN1

rearrangement begins at the

B-chain locus (chromosome 7) 

sometimes y-chain rearrangement occurs in parallel 

• cell: DN2

B-chain completes recombination and forms an

in-frame protein

• cell: DN2

how many attempts can be made to produce a functional B-chain

4 attempts

• TCR B locus contains two separate clusters of DB, JB and CB segments (DB1-JB1-CB1 and DB2-JB20-CB2)

• each allele can attempt rearrangement in both clusters - giving 4 total chances (2 alleles x 2 clusters) 

• if one rearrangement fails (out of frame), the thymocyte can try again on the other cluster or allele before apoptosis