T Cell Development

Question 1

T cell development in the thymus

Answer 1

• the major but not exclusive site, for the maturation of T (thymus-derived) cells
• site of TCR gene rearrangements
• provides the appropriate microenvironment for the maturation of precursors and their selection (positive and negative selection) based on their TCR specificities
• site of ordered stages of maturation that have been phenotypically identified and functionally committed

Question 2

The Importance and Function of Thymus

Answer 2

• immunodeficiency associated with defects in T cell function caused by:
• Neonatal thymectomy (1952- JFAP Miller)
• DiGeorge’s Syndrome (human selective T cell immunodeficiency- epithelial defect)
• Natural (spontaneous) mutations- nude mouse a defect due to the loss of a TF involve in thymic epithelial cell differentiation
• only about 3% of the thymocytes ever leave the thymus- a site of maturation, cell proliferation, and cell death (by apoptosis)
• very active in fetal and early life, it later undergoes involution but can be re-activated as a site of thymocyte development in the older individual

Question 3

Embryonic thymic development

Answer 3

• in the fetus, the thymus is formed principally from the fusion of the 3rd pair of pharyngeal pouch (endoderm) and cleft (ectoderm)
• mesenchyme surrounding the epithelial components condense to form the capsule. The mesenchyme also penetrates the thymic rudiment for form the trabeculae
• hematopoietic precursors seed the thymic rudiment beginning at 8 weeks of gestation

Question 4

Cellular organization of the thymus

Answer 4

-the thymus has a capsule, and a subcapsule region
-there is a trabeculae that go down into it
-there is a cortical epithelial cells at the top
-and then there is corticomedullary junction
and then it goes to the medulla

Question 5

Immigration and Emmigration of Thymus

Answer 5

• T cell precursors (prothymocytes) derived from fetal liver and bone marrow seeds the thymus
• thymic anlage produces chemotactic factors that attract T cell progenitors
• cells that seed the thymus enter at the cortico-medullary junction via blood vessels
• thymocytes that survive the rigors of selection leave the thymus from venules in the medulla- also controlled by chemokines and sphingosine 1-phosphate receptors

Question 6

FACS analysis of thymocyte

Answer 6

• 2% double negatives, which are most immature
• then 88% double positives
• then about 10% mature cells either CD4+ or CD8+

Question 7

Double Negative Cells

Answer 7

• CD4-CD8- cells 2-5% total thymocytes
• contain least mature cells, considerable cell division
• 2/3rds are triple negative based on TCR expression (lack CD3)
• TCRbeta,gamma, and delta rearrangement occurs at this time
• 1/3 are mature TCRgammadelta+ and TCRalphabeta+ cells

Question 8

Double Positive Cells

Answer 8

• CD4+CD8+ cells: 80-85% of total thymocytes
• TCRalpha rearrangement occurs here
• most have rearranged TCRalpha and beta genes and express low levels of TCR
• small subset has high levels of TCR (most mature, positively selected cells)
• small subset is actively dividing (earliest DPs)
• most apoptosis occurs here, very sensitive to apoptosis inducing agents, especially sensitive to glucocorticoids

Question 9

Single Positive Cells

Answer 9

• CD4+CD8- and CD4-CD*+ cells: 10-15%
• most are mature cells with high levels of CD3 and TCRbeta
• CD4:CD* approx 2:1
• most SP cells are functionally mature and destined to leave the thymus
• small set of SPs are immature (ISP)- have low CD3 and TCRbeta transitional cells that are on the way from DN to DP

Question 10

T cell developmental pathways

Answer 10

• CD4-CD8-:
• get to thymus
• delta, gamma, beta rearrangement
• split to either rearrange beta, or rearrange delta and gamma
• the delta gamma are done at this point since they don’t have MHC
• the beta is then considers a preTCR

CD4+CD8+:
-the preTCR goes through alpha rearrangment

Single postive:
then the alpha beta is class I restricted (CD8+)
or class II restricted (CD4+)

Question 11

Two checkpoints involved in alphabeta T cell development

Answer 11

• First checkpoint: mediated by pre-TCR (also referred to B-selectin)
• can’t go passed if don’t have RAG, CD3E, TCRBeta, pTalpha

-Second checkpoint: mediated by mature alphabeta TCR (also referred to as positive and negative selection)
-can’t go pass if don’t have TCRalpha, MHC II (CD4),
Beta2 microglobulin (CD8), bare lymophocyte syndrome

Question 12

Checkpoint 1

Answer 12

-Purpose: assesses whether TCRbeta chain is functionally arranged

Consequences:

• allelic exclusion at the TCRbeta locus
• proliferation
• expression of CD4 and CD8
• termination of transcription at TCRgamma locus
• initiation of rearrangments at the TCRalpha locus

What stimulates the pre-TCR to signal?

• no known ligand
• pre-TCR can mediate signal without ectodomains of pTalpha and TCRbeta

Question 13

Checkpoint 2

Answer 13

Purpose: Assesses 1) whether TCRalpha is functionally rearranged; 2) whether TCR is self MHC-restricted; 3) whether the TCR is auto-reactive

Consequences: 1) maturation of thymocyte to functionally competent SP cell
2) Establishes a self-MHC restricted, non-autoreactive TCR repertoire with appropriately match co-receptors and functional potential

-requires peptide/MHC molecule interactions to induce a signal

absence of interaction leads to apoptosis

distinct positive vs negative selection signals

Question 14

Ligands for positive and negative selection

Answer 14

• ligands are the peptide/MHC complexes expressed on stromal and hematopoietic cells. Peptides are derived from endogenous sources in the thymus or serum components
• it is critical to delete thymocytes expressing TCRs that would react with BM-derived APCs in the periphery. Therefore, presentation of self-endogenous peptides from hematopoietic cells plays an important role in shaping the repertoire
• what about peripheral tissue specific antigens? Recent work on the Aire transcription factor suggests it plays a key role in regulating the expression of some tissue specific antigens in medullary epithelial cells in the thymus. Absence of Aire results in specific types of autoimmunity in man and mouse

Question 15

Engagement of the TCR by self MHC can lead to further maturation and to cell death

Answer 15

• avidity depends on the affinity of the TCR-peptide/MHC interaction and the density of the peptide/MHC on the thymic epithelial cell
• avidity determines the strength of signal delivered which dictates the outcome
• stronger signals may mean longer signaling or additional signaling
• too little: cell death, too much: cell death

Question 16

Site of positive selection

Answer 16

• cortical epithelial cells

- differential expression of MHCII using different promoter transgenics

Question 17

Site of negative selection

Answer 17

• can occur in cortex or medulla, depending on where high avidity interactions occurs. But normally negative selection is likely to be concentrated at cortico-medullary junction and in medulla
• see increased autoreactive CD4+ T cells if medulla lacks class II MHC molecules
• tissue specific autoimmunity develops if peripheral antigens are not expressed in thymic medually epithelial cells (Aire mutation)
• cell types that can induce negative selection: thymic DCs, cortical epithelial cells, medullary epithelial cells

Cannot: thymic macrophages( they just eat)