T Cell Development Flashcards
What is the goal of T cell development?
generate a mature pool of T lymphocytes, each w a single specificity, that recognises foreign but not self antigen when complexed w self MHC molecules
– self tolerant and self restricted
challenge
– Ag recognition occurs via a specific TCR; generated by random rearrangement of segmented genes
– not all arrangements are productive, many of the TCRs don’t recognise self MHC plus peptide and some will be self-reactive
What evidence shows that T cell development occurs in the thymus?
Nude mice lack a functional thymus due to a mutation in the Foxn1 gene, a TF which is essential for thymus development
this results in the absence of mature T cells, and the mice are severely immunocompromised
– able to accept skin graft from other mice, and even rats
– no cell mediated immunity, no Ab response to most Ags
What are the components of the thymus?
defined by histology: cortex and medulla
stroma composed of BM derived and non-BM derived elements
epithelial component of stroma provides specialist functions (cTEC and mTEC)
– cortical and medullary thymic epithelial cells
macrophages clear dead cells
hassall’s corpuscles: secrete TSLP - activated thymic DCs which induce and generate Tregs
How does colonisation of the thymus occur?
there is no self-renewing intrathymic lymphocyte population under normal conditions
– colonisation thought to occur in waves
- vasculature-independent (fetal thymus prior to establishment of vascular network)
– migration through connective tissue, mediated by chemokines eg CCL21 and 25 - vasculature dependent (late fetal and postnatal thymus)
– entry at cortico-medullary junction, mediated by chemokine receptors eg CCR7 (CCL19 and 21) and CCR9 (CCL25)
– requires seeding cells to adhere to P-selectin glycoprotein (PSGL1), V-CAM, and I-CAM on thymic endothelial cells
What are the seeding cells?
the progenitor cells that migrate from the bone marrow to the thymus to initiate the development of T cells
phenotype of earliest intrathymic progenitors, early thymocyte progenitors (ETP)
- cKit+ within DN1 population
- Flt3+ cells within ETP gate shown to be canonical T cell progenitor
- single Flt3+cKit+ ETP can generate T, B, GM, and macrophages in in vitro assays
- EPT most closely related to lymphoid-primed multipotent precursor (LMPP)
Outline the commitment in T cell development.
progress down T cell pathway begins when DN1 (CD44+25-) cells make a transition to DN2 stage (CD44+25+)
– DN2 can still make macrophages, NK cell, DC in vitro
non-T cell fate choices lost by DN3
How does Notch regulate gene expression?
family of TM receptors (1-4)
5 ligands (mammals)
– delta-like 1, 3, 4
– jagged 1, 2
upon ligand binding, intracellular portion of Notch, ICN, is proteolytically cleaved and translocated to nucleus -> interacts w CSL (RBPJk) which binds to co-factors to induce gene transcription
What experiments show that Notch is critical in T cell development?
creation of Notch I knockout using Cre-loxP system
– cross w MxCre mice; Cre expression induces in response to IFNa
conditional deletion of Notch I abrogates intrathymic T cell development
– specific block in T cell development at DN1 stage
– all cells end up becoming B cells as shown by B220 expression
What cells produce ligand for Notch1, and what evidence shows this?
delta-like 4 and delta-like 1 produced by cTECs and mTECs
FoxN1 deletion in only TECs
- again, progenitors become B cells as they cannot enter T cell development
How do Notch, GATA3, and TCF7, induce commitment to T cells?
TFs GATA3 and TCF1, both required for T cell development
GATA3 represses B cell lineage potential in ETPs
TCF7 mainly acts as a positive regulator of T cell specification
Notch, GATA3, and TCF7 upregulate expression BCL11B
– allows full commitment to T cell lineage as it is expressed during DN2 (specifically DN2b)
What is beta-selection?
a checkpoint during T cell development at DN3
expression of surrogate light chain and formed heavy chain (pre-TCR) on cell surface signals through CD3 and rescues cells from apoptosis and initiates further development
If preTCR binds p:MHC, proliferation of DN3 is enhanced
Along with Notch, and the pre-TCR, what other receptor is required at the beta-selection checkpoint?
CXCR4 binding SDF-1α (aka CXCL12)
causes downstream signalling which upregulates BcI-2A1, inhibits caspase 3-mediated apoptosis
What happens after beta-selection (formation and signalling through pre-TCR)?
failure to pass beta-selection (or make gamma-delta receptor) causes death by apoptosis
after b-selection:
– lose pre-TCR
–DN4 cells undergo a burst of proliferation, then upregulate CD8 and CD4 = DP
DP then enter quiescent phase
– Rag genes reactivated
– Valpha genes begin to rearrange
TCR alpha loci undergo successive rearrangements until cell is positively selected or die
What are two models of TCR receptor lineage commitment?
Stochastic selection
- cells pre-committed to alpha-beta or gamma-delta before TCR rearrangement
- cells that develop wrong receptor die
signal strength
- ligand engagement of the gd-TCR elicits strong signals that result in commitment to this lineage
- transduction of weak signals can result in commitment to ab-TCR lineage and the generation of DP thymocytes
– this is the favoured model
How does the thymic stroma regulate thymocyte development?
- early stages take place in the cortex
- thymocytes at different stages are found in different intracortical organs
- outward migration can be partly mediated by CXCL12/CXCR4 and CCL19/21 - CCR7
- but still not understood
What is the role of cTECs?
- T cell lineage commitment -DL4
- thymocyte differentiation
- intrathymic T cell migration
- positive selection (express MHCI and II)
- no major role on negative selection
What percentage of DP cells leave the thymus after positive and negative selection?
only <5%
highly selective process that ensures:
- exported T cells are useful
- to remove T cells that are harmful
What is positive selection?
occurs in thymic cortex
- selection of TCRs that can binds MHC:peptide
- selection for class I or class II reactivity
why?
1. gene rearrangement is versatile but inherently easteful (~30% chance of being in frame)
2. T cells need to interact w p:MHC on other cells, not free Ag
3. functional specialisation of cells expressing class I and II MHC restricted TCRs is distinct
DP thymocytes that cannot binds self MHC:peptide complexes die by apoptosis (death by neglect ~95%)
What evidence shows MHC is an essential component of positive selection?
TCR transgenic thymocytes are only selected by the MHC allele to which they are restricted
in the absence of the selecting MHC, thymocytes remain in the cortex and fail to mature
What role do cTECs play in positive selection?
they express class I and class II MHC
but lacks CD80/86
express thymoproteasome
Is there a role of peptide in positive selection?
TAP-1 is a part of the TAP complex required for peptide loading into MHC class I in the ER
in the absence of TAP-1, CD8 selection is markedly impaired
What is the thymoproteasome?
proteasome involved in delivering peptides for loading into class I MHC
inactivates regulatory proteins and remove aberrant proteins by degrading them in combination w ubiquitin system
expresses novel catalytic subunit of the proteasome, beta5t (different from beta5i in immunoproteasome)
expressed exclusively in mTECs
Does expression of beta5t influence positive selection?
beta5t differs from beta5i in that it produces a pocket that is mainly hydrophobic rather than hydrophobic
– gave rise to prediction that thymoproteasome may have weaker chymotrypsin activity than the conventional proteasomes, so may produce different peptides
beta5t KO mouse had v marked reduction in CD8 SP thymocytes
– CD4 SP thymocytes OK
What does positive selection require?
- a peptide and beta2M (MHC)
- beta5t - special thymic peptides
- no. of maturing CD8+ T cells was dependent on the complexity of the added peptides
What is the nature of the peptide that drives self tolerance during negative selection?
agonist peptide -> deletion
– v strong binding
antagonistic peptide -> selection
– weak or moderately binding
the difference in positive and negative selectors is how tightly they binds to the receptor
CD8 T cells are fully dependent on the presentation of peptides from the thymoproteasome, how are CD4 T cells selected?
cathespin L KO mice have defective selection
- 60-80% decrease in CD4 SP
TSSP (tissue specific serine protease) KO mice have normal CD4 number but altered repetroire
How does commitment of DP thymocytes to the CD4 or CD8 SP lineages occur?
entirely based on TCR engagement, no cytokines involved
reflects ability of TCR to recognise peptides bound to class I or class II MHC
What are the factors that determine lineage commitment?
more Lck associated more w CD4 than CD8
– evidence suggests Lck signalling affects cell fate
– 50% of CD8a polypeptides (part of co-receptor) in thymus so not have a cytoplasmic domain so cannot associate w Lck
kinetic signalling model
– SP arise through intermediate, CD4hiCD8lo
– to become CD4 SP, cells need continuous signalling, while CD8 SP arise because here is a break in signalling
What are the key transcription factors that direct CD4 commitment?
– Th-POK is upregulated in response to strong TCR signaling with MHC class II
– Th-POK activates genes necessary for CD4⁺ T cell identity and represses Runx3, which is needed for CD8 lineage
– GATA 3 is also involved, but KO do not redirect to CD8 lineage unlike Th-POK KO
– enforced GAT-3 expression does not redirect
MHC class I restricted thymocytes to CD4 lineage
– GATA3 induces ThPOK expression (w help)
What are the key transcription factors that direct CD8 commitment?
Stat5 and Ets1 contribute to Runx3 expression
–IL-7 is essential for thymocyte survival and development
– Upon IL-7R engagement, JAK kinases phosphorylate Stat5 -> dimerization and translocation to the nucleus -> transcription of Runx3
– Ets1 binds to adjacent sites on the Runx3 promoter or enhancer regions, stabilizing the transcriptional activation
Runx3 is TF that is upregulated in response to weaker TCR signaling with MHC class I
– Runx3 promotes the expression of genes that drive CD8⁺ T cell identity and suppresses Th-POK
What is negative selection?
occurs in thymic medulla after positive selection
involves removal of cells w capacity to respond to self-peptide
- those that do = death by apoptosis
How does negative selection occur?
strong signals through the TCR drives cell into apoptosis rather than division
negatively selecting cells express CD80 and CD86 so give co-stimulation
strong TCR signal induces high Ca2+ and PKC activation rather than calcineurin
PKC induces expression of pro-apoptotic Bcl-2 family member Bim
- narrow window between positively and negatively selecting outcomes
What is AIRE?
a TF expressed in mTECs, that allows expression of tissue-specific antigens form different organs
– main method of central tolerance allowing negative selection of self-tissue reactive T cells
– exact number unknown but may regulate 1000s of genes
What is another TF involved in expression of self-peptides?
Fezf2
not as important as AIRE, as it only acts on open chromatin, whereas AIRE can act on closed chromatin
How do Tregs arise?
TCR signals that are strong but not strong enough to induce apoptosis causes upregulation of IL-R2 (CD25)
DCs within the thymus produce IL-2
– IL-2 signalling induces upregulation of FoxP3
= Tregs
existing thymic Tregs restrict production of more Treg by outcompeting for IL-2
What are the key events in each section of the thymus?
cortex
- positive selection = peptide:MHC recognition
medulla
- negative selection (can occur in cortex too) = self-peptide
- lineage commitment: CD4 or CD8
- recognition
- Treg differentiation
- maturation (including proliferation)
