development of T lymphocytes

what is lymphopoiesis

production of new lymphocytes in central lymphoid organs

what is the thymus

primary site of t cell development

what happens to t cells in the cortex of the thymus

t cells develop and proliferate 

what happens to t cells in the medulla of the thymus

mature t cells are selected, autoreactive t cells are eliminated

what is the thymic stroma in the thymus

cortical epithelial cells and medullary epithelial cells

what is the role of thymic stroma in thymus

structure and environment for t cell development

in a young adult mouse, the thymus contains how many thymocytes

10^8

in a young adult mouse, how many thymocytes generated

10^7

in a young adult mouse, how many thymocytes leave the thymus each day

10^6 (2-4%)

in a young adult mouse, how many thymocytes die by apoptosis

98%

what are the key players in apoptosis

macrophages in thymic cortex and medulla

why is the loss of thymocytes important for T cell development 

each thymocyte is carefully tested to make sure it can recognize self-MHC with self-peptides and that it wont react strongly against the body’s own tissues (self tolerance)

what marks the successive stages in thymocyte development

• status of TCR genes

• changes in TCR expression

• changes in cell-surface molecule expression 

particular combos of cell-surface proteins are used as markers for T cells at ___

different stages of differentiation 

two distinct lineages of cells

1. gamma delta T cells

2. alpha beta T cells (develop into CD4 and CD8 T cells)

commitment to T cell lineage occurs in the thymus after ___

notch signalling

what is notch signalling

notch (transmembrane receptor) binds to ligand and triggers cleavages

in the thymus what are early thymic progenitors

CLPs (common lymphoid progenitors) aka DN1

role of ETPs and TECs in notch signalling

ETPs receive notch signaling from TECs (essential for T cell lineage commitment, required during early phases of thymocyte differentiation up to the DN3 stage)

what happens during B-selection (DN4)

notch signaling is turned off as a consequence of pre-TCR signaling

features of T cell precursors in the thymus

• have not rearranged TCR loci

• do not express CD4 or CD8

• called double-negatives

markers of t cell precursors in the thymus

• c-KIT: receptor for stem cell growth factor 

• CD44: adhesion molecule homing to thymus

• CD25: alpha chain of IL2 receptor

NOTCH functions/features

• become a T cell

• expressed early in DN1-DN3

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

• without notch signaling, thymocytes fail to adopt the T cell lineage 

CD3 features/functions

• signal through your TCR

• appears at DN to DP transition

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

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

IL-7R functions/features

• stay alive and divide

• expressed in early DN (DN1-DN3)

• provides survival and proliferation signals during early thymocyte growth

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

RAG-1/2 functions/features

• build your TCR

• active during DN2-DN3 and again at DP

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

• 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

• stops further B chain rearrangement (maintains allelic exclusion)

• triggers TCR a-chain rearrangement 

• promotes transition to CD4+CD8+ DP stage 

what is allelic exclusion

recombination shutdown, prevents further recombination of B chain

why is allelic exclusion vital

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

key point of pre-TCR

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

in T cell development in thymus, what happens after B chain rearrangement is complete

DN3 cells progress to DN4

what happens when a cell progresses to DN4

CD4 and CD8 are expressed (cells are double positive (DP))

what does expression of CD4 and CD8 initiate

rearrangement of a chain locus in double positive cells

what happens in the rearrangement of a chain locus in DP cells

a chain pair with already functional B chain, forming complete aBTCR on cell surface

what marks the start of selection process

at DP stage, TCR and associated CD3 complex are expressed at low levels, just enough for cell to test whether the receptor can recognize self-MHC molecules presenting self peptides

what is selection process

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

in the thymus, what is mainly found in the outer cortex

DNs

in the thymus, what is mainly found in the deeper cortex

DPs

in the thymus, what is mainly found in the medulla

SPs

in the thymus, what is mainly found in the cortical epithelial cell (cTECs)

express MHC and MHC II (role in positive selection)

in the thymus, what is mainly found in the medullary epithelial cell (mTECs)

present peripheral antigens (role in negative selection)

stages of gene rearrangement in aB T-cells: germline gene configuration

both TCR a and B loci are in germline (unrearranged form), cell: early DN1

stages of gene rearrangement in aB T-cells: DB to JB rearrangement

rearrangement begins at the B-chain locus (chromosome 7). sometimes Y-chain rearrangement occurs in parallel. Cell: DN2

stages of gene rearrangement in aB T-cells: VB to DJB rearrangement in frame 

B-chain completes recombination and forms an in frame protein, cell: DN2

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

up to 4

why can there be 4 attempts to form a functional B-chain

• TCR B locus contains 2 separate clusters of DB, JB, and CB segments 

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

what happens if one rearrangement fails (out of frame)?

the thymocyte can try again on other cluster or allele before apoptosis

what does the redundancy of 4 attempts to produce functional B-chain increase

chance of producing a productive B-chain before cell dies

stages of gene rearrangement in aB T-cells: VB to DJB rearrangement in frame, B-chain protein produced

B protein pairs with surrogate a-chain (pTa) to form pre-TCR. cell: DN3- intracellular B protein detected

stages of gene rearrangement in aB T-cells: surface expression of B chain with surrogate a chain, B rearrangement stops, cell proliferates

pre-TCR moves to surface, induction of CD4 and CD8 expression (DP stage), cell: DN4

stages of gene rearrangement in aB T-cells: Va to Ja rearrangement

occurs at a-chain locus once B is fixed. functional aBTCR and CD3 appear at the surface. cell: DP- ready for selection (positive/negative)

what does the arrangement of a-chain locus allow

sequential recombination.

• a-chain has multiple Va and Ja gene segments arranged linearly

• if a rearrangement is nonfunctional, the cell can try again using different Va and Ja combinations upstream or downstream

sequential recombination continues until:

1. thymocyte successfully forms a functional aB TCR that can recognize self-MHC (positive selection) 

OR

2. it fails repeatedly and dies by apoptosis 

stages in T-cell development in the thymus

• if positive rearrangement is made, a aB TCR is expressed on thymocyte surface

• cells then undergo positive and negative selection

• cells that fail selection undergo apoptosis

• those that pass the selection step lose either CD4 or CD8, becoming single positives 

• mature SPs leave the thymus

3 checkpoints of T-cell development

1. TCRB rearrangement (B-selection)

2. TCRa rearrangement and positive selection

3. negative selection

goal of positive and negative selection of T-cells

to produce T cells that recognize foreign antigens presented on self-MHC molecules, but ignore self

positive selection

• occurs in thymic cortex

• keeps only T cells that can bind to self-MHC (class I or II)

• results in MHC restriction

negative selection

• occurs in both cortex and medulla

• removes T cells whose TCR bind self-peptide: self-MHC too strongly

• these cells die by apoptosis in thymus

• results in self tolerance

in affinity strength of too cold, what happens to TCR-MHC interaction and outcome

• no or very weak binding

• no selection, death by neglect

in affinity strength of just right, what happens to TCR-MHC interaction and outcome

• moderate binding

• positive selection, MHC restriction

in affinity strength of too hot, what happens to TCR-MHC interaction and outcome

• strong binding to self peptide: self-MHC

• negative selection, apoptosis

in affinity strength of alternative selection, what happens to TCR-MHC interaction and outcome

• intermediate/unique affinities

• may lead to nonconventional T cells

negative selection within the thymic medulla

negative selection of mature SP thymocytes occurs to self antigens presented on APCs (mTECs and DCs)

what does AIRE (autoimmune regulator) do

enables thymic epithelial cells to undergo promiscuous gene expression, enables presentation of tissue-specific (tissues outside of thymus), self-antigens (or peripheral antigens) from across the body (e.g., insulin, myelin)

key point of AIRE

lets thymus show T cells self-antigens from across the body, ensuring self reactive cells are deleted, central tolerance

what induces thymic emigration

sphingosine 1-phosphate (S1P) receptor

steps of thymic emigration

after completing selection in the medulla, mature CD4+ and CD8+ T cells upregulate the receptor S1PR1, which senses S1P gradients into blood and lymph, guiding their exit into circulation

where is S1P present in high concentrations

blood and lymph

what does S1PR1 act as

GPS, senses high levels of S1P in blood to direct mature T cells out of the thymus