T-Cell Maturation & Differentiation

Question 1

Describe the thymus as an organ. When does it develop. What cells “home” to the thymus? What cells will develop here?

Answer 1

The thymus is an epithelial-lymphoid organ that develops very early during embryonic
development. Hematopoietic stem cells (HSC) home to the thymus and develop into: T helper (CD4), cytotoxic (CD8), natural killer T (NKT) and T regulatory (Treg) cells.

Question 2

How does the epithelial component of the thymus develop? When during gestation?

Answer 2

The epithelial component of the thymus derives bilaterally from epithelium of the third
pharyngeal pouch at fourth week of gestation.

Question 3

What happens in regards to the thymus between the 4-7th week of gestation?

What happens during the 12th week?

Answer 3

Between 4-7th week of gestation, the primordial
thymic glands lose connections with the pharynx and migrate to the final position in the
mediastinum forming a single bilobate gland.

7th week- colonization by HSC
12th week- begin to produce T cells at 12-13th week, mature T cells egress the thymus at 13-14th week
p 2

Question 4

The normal human thymus develops early on in fetal development;

When are glands colonized by HSC?

When does thymus begin to produce T cells?

What happens at the end of 13th week and beginning of 14th week?

What is the the end result?

Answer 4

the glands are colonized
by hematopoietic stem cells at 7-8 weeks of gestational age.

The thymus begins to produce T cells around 12-13 week of gestation.

Mature T cells egress the thymus and colonize peripheral lymphoid organs at the end the 13
and the beginning of the 14 week of gestation.

Thus, by the time the baby is born, the peripheral T cell repertoire is established to the
point that thymectomy does not cause immediate immune deficiency.

Question 5

What causes DiGeorge Syndrome (DGS)/ Velo-Cardio-Facial Syndrome (VCSF)?

Describe the disease. What might symptoms include? Why recurrent infections occur?

How may it be treated?

Answer 5

DiGeorge syndrome is caused by a large deletion in chromosome 22 which is caused by an error
in recombination at meiosis.

• It is a rare congenital (i.e. present at birth) disease syndrome whose symptoms vary greatly between individuals but commonly include a history of heart defects, characteristic facial
  features and recurrent infection due to absence of the thymus and T cells.

The absence of the functional thymus results in complete T cell deficiency and severe
immunodeficiency.

Transplantation of an allogeneic thymus graft into patients with DGS rescues T cell
deficiency.

Question 6

What are the implications for a Mutation in the FOXN1 gene:

What results?

What type of treatment might help?

Answer 6

The FOXN1 gene (on chromosome 17) encodes a transcription factor that is essential for the functional maturation of thymic epithelial cell progenitors.

In these patients, the thymus gland fails to form in utero because epithelial progenitor cells fail to undergo functional differentiation and instead form cyst-like structures with immature morphology.

The immature epithelial cells fail to recruit hematopoietic stem cells into the organ.

Thus, the functional maturation of thymic epithelial cells is required for the development
of a normal thymic architecture, which is essential for the production of various thymicdependent T cells subsets and the initial establishment of the peripheral T cell pool in
animals and humans.

• Thymic implant recently been shown to restore T cell immune response in patients with
  Foxn1 mutations

Question 7

Describe the cellular composition of the thymus glands.

What does the thymic stroma include?

Answer 7

See p 4 
capsule: fibroblasts
cortex: developing T cells
medulla- mature T cells
cortical-medullary junction

Thymic stroma: this includes the predominant thymic epithelial cells (TEC) and fibroblasts.

a. Fibroblasts: found in the thymic capsule and septa.
b. Thymic epithelial cells (TEC): provide three critical functions for the development of T
cells.

Question 8

Thymic epithelial cells (TEC):

From where are they derived?

Provide three critical functions for the development of T
cells. Describe.

Answer 8

All TEC are derived from
endoderm.

TEC produce cytokines such as IL1, IL6, IL7, and SCF (stem cell factor), TSLP
(thymic stroma lymphopoietin) that are requires for growth and differentiation of
various immature T cells.

TEC also express cell surface molecules such as ligands Delta-like-4 (DL-4) and DL-1 for the notch receptor. Signal of notch receptors expressed on progenitor cells is required for T cell lineage commitment.

TEC expression of MHC classes I, and II/self antigen complexes controls the
selection of maturing T cells.

Expression of peripheral tissue antigens: for example insulin

Question 9

Describe macrophages and dendritic cells.

Where do they mature?

Where are they located?

Function?

Answer 9

these cells mature from the bone marrow and migrate into the thymus. These cells are scattered in the cortex and medulla; however, they highly populate the cortical-medullary junction. They function: in antigen presentation; deletion of
autoreactive T cells (negative selection) and phagocytosis of apoptotic thymocytes.

Question 10

Describe thymocytes:

How are thymocytes derived?

Describe the different types of T cells the thymus is responsible for?

Answer 10

The predominant lymphoid cells in the thymus. In postnatal animals,
thymocytes are derived from progenitor cells of the bone marrow, the hematopoietic stem cells (HSC). After arriving in the thymus, bone marrow HSC progress through tightly
regulated steps to develop into mature T cells. The thymus is responsible for the
development of four functionally different T cells: CD4 T helper, CD8 T cytotoxic,
regulatory T cells (Treg) and natural killer T cells (NKT).

Question 11

How can flow cytomettric analysis be used in regards to T cells? Describe process.

Answer 11

Flow cytometric analysis is a powerful tool to identify thymocyte subsets: cells stained with anti-CD4 and CD8 antibodies are separated into 4 distinct populations: DP, double positive (CD4 and CD8 positive); DN, double negative (either CD4 or CD8 is expressed).

Question 12

Draw a flow chart for the early stages of human T cell development.

Answer 12

p 6

The thymus is populated by blood born progenitor cells that are derived from the bone marrow
hematopoietic stem cells (HSC). These cells express the unique cell surface marker CD34 and
have the capacity to develop into T cells as well as B cells, dendritic cells, and NK cells.

Upon entry into
the thymus, the lineage potential of the CD34pos cells is restricted to only the T lineage.

Question 13

Why can cord blood be a good source for HSC CD34pos cells for transplant

Answer 13

Because the migration of CD34pos cells is initiated at 7-8 week of gestation and is highly active in the
neonatal period, cord blood is good source for HSC CD34pos cells for transplant.

Question 14

How does T cell production by thymus change with age.

Answer 14

The production of T cells by the thymus is declines with age; This age-associated decline in the production of T cells is responsible for the decline in immune response in the elderly.

Numbers given p 7

Question 15

What are the four developmental events in T cells. (List)

Answer 15

• T lineage commitment
• Proliferation and differentiation
• Selection: positive and negative
• Maturation

Question 16

When is a T cell committed to T lineage?

Answer 16

See chart p 8

Rearrangement of gamma, delta beta

Pre-T with CD34 and CD1A pos

Question 17

Discuss the significance of the Notch receptor signal.

Answer 17

The Notch receptor signal is essential for T cell lineage commitment of the CD34pos
HSC. Signal through the notch receptor terminates the potential to commit to B and myeloid lineages (monocytes and DC). The cells have potential to become T or NK cells
(T/NK). Persisting Notch signaling terminates NK development.

Question 18

What happens in T cell development after the notch signal by Notch ligand DL4 and DL1?

What factors/enzymes will be needed?

Answer 18

cells commit to T lineage,
express CD1A and begin to rearrange TCRgamma,delta and beta genes. Cells at this stage, pre-T, can
develop into either TCRgamma/delta or TCRalpha/beta T cells. Te rearrangement of TCR genes requires the recombinant activation gene -1 (RAG1) and RAG2. IL7 is also needed for this developmental stage.

Question 19

After the notch signal by Notch ligand DL4 and DL1 cells commit to T lineage,
express CD1A and begin to rearrange TCRgamma,delta and beta genes. Cells at this stage, pre-T, can
develop into either TCRgamma/delta or TCRalpha/beta T cells.

Describe what happens in the next stage.

Answer 19

In the next stage, cells express CD4 and become immature single positive (CD4ISP). These cells express the preTalpha (pTalpha); the expression of pTalpha also induces the expression of CD3.

The pT-alpha together with CD3 and TCR-beta forms the preTCR complex. Signaling via pre-TCR generate a weak activation of the extracellular signal related kinase (ERK)
which terminate rearrangement of TCR-gamma and TCR-delta genes.

Question 20

What composes the pre-TCR complex?

What does expression of pre-TCR complex allow for?

Answer 20

The pT-alpha together with CD3 and TCR-beta forms the preTCR complex.

The expression of the preTCR allows the ISP cells to undergo the Beta
selection process

Question 21

The expression of the preTCR allows the ISP cells to undergo the Beta
selection process. Describe the Beta selection process.

What two things could happen at the end of this process?

Answer 21

This process selects cells with productive and functional rearranged
TCR-beta genes signaling via the preTCR. Cells that do not from a functional preTCR die by apoptosis. Cells that survive the Beta selection step proliferate and expand.

This enormous expansion of cells at this stage is responsible for generating the large number of thymocytes with TCR-alpha/beta in the thymus. Expression of CD3 is up-regulated at this stage.

Question 22

Cells that survive the Beta selection step proliferate and expand.

What happens to ISP cells that are selected to progress?

Answer 22

ISP cells that are selected progress to express CD8 and thus are termed CD4 CD8 double
positive cells (DP); these cells begin to rearrange TCR-alpha genes. Because the TCR-delta locus
is within the TCR-alpha genes, rearrangement of the TCR V-alpha genes will delete the delta locus.
There is no allelic exclusion in TCRV-alpha gene rearrangements. It is possible that there are two different rearranged V-alpha chains, each is associated with a common V-alpha. 

Subsequent positive selection will ensure that each T cell only has a single functional specificity

Question 23

How is it determines whether a T cell becomes CD4 or CD8?

Answer 23

Depend on the signal generated via the interaction of the co-receptor CD4 or CD8 surface
molecules, DP cells down modulate one of the co-receptors and mature into either single
CD4 or CD8 T cells.

p 10

Question 24

Describe the positive selection of TCR-alpha/beta T cells: Selection of T cells that recognize antigens presented by MHC molecules.

Answer 24

The DP thymocytes with functional TCRalpha-beta/CD3 complex must recognize self-peptides
in the MHC I and II complex presented by cortical TEC.

• The interaction of TCRalpha-beta/CD3 complex with self peptides and MHC antigens must be of low affinity; DP cells with low affinity TCR for self peptides are rescued from apoptosis and proliferate (1 or 2 rounds of replications).

These cells shut down expression of
recombination-activating gene (RAG); no further rearrangement can take place if RAG
proteins are not present.

Cells express TCRalpha-beta that do not recognize self peptides undergo apoptosis.

Positive selection skews the selected TCRalpha-beta repertoire toward self peptides and the
potential of generating autoreactive T cell clones.

Question 25

What happens with patients that receive bone marrow transplants?

Answer 25

Patients who receive bone marrow transplant generate T cells that recognize foreign antigens in the context of their own MHC antigens and not donor MHC, indicating that developing donor T cells occurs on TEC of the patient recipient.

Question 26

Describe negative selection. (What is it/what does it result in?

Answer 26

Deletion of mature T cells that bind strongly to MHC/antigens (autoreative T cells); negative selection results in the generation of central tolerance.

Question 27

Negative Selection: Deletion of mature T cells that bind strongly to MHC/antigens (autoreative T cells); negative selection results in the generation of central tolerance. Describe this process. Where does it primarily occur?

Answer 27

DP cells with TCRalpha-beta/CD3 complex with high affinity for self-peptide and MHC are instructed to undergo apoptosis; this process is necessary to eliminate autoreactive T cells and to establish the central tolerance. Negative selection occurs predominantly in the cortical-medullary region where a high density of thymic DC cells is present. The DC cells are also responsible for phagocytosis of apoptotic cells.

Question 28

What is AIRE and what does it do?

Answer 28

TEC also participate in clonal deletion of T cells that are reactive to organ specific antigens. The Auto Immune Regulator Element gene (AIRE) encodes a transcription factor that induces expression of a battery of peripheral-tissue antigens by thymic medullary epithelial cells; thus, AIRE promotes central tolerance of thymocytes by inducing negative selection, contributing to the prevention of organ specific autoimmunity.

Question 29

Describe disease associated with mutations of AIRE.

Answer 29

AIRE mutations are responsible for the development of autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) or Autoimmune polyendocrinopathy syndrome type 1 (APS1). The affected organs include: adrenal, thyroid, parathyroid, and pancreas.

Question 30

What happens to DP cells that survive both the positive and negative selection processes? Where do mature CD4 and CD8 cells reside?

Answer 30

DP cells that survive both the positive and negative selection processes will commit to either single CD4Pos or CD8Pos cells. - Selection with CD4 co-receptor and MHC-II will generate mature CD4 cells. - Selection with CD8 co-receptor and MHC-I will generate mature CD8 cells - Mature CD4 and CD8 cells are predominantly resided in the medulla of the thymus. - Mature CD4 and CD8 cells egress the thymus via blood vessels in the septa in the cortical-medullary junction.

Question 31

Describe the development of TCR gamma/delta cells.

Answer 31

develop from the ISP cells, the exact mechanism for the lineage choice between TCRalpha/beta and TCRgamma/delta is not well understood. It is generally accepted that the expression of pT-alpha and signaling of this receptor skews the cells toward TCRgamma/delta lineage.

Question 32

Describe TCR gamma/delta cells. How prevalent are they? Where are they? What type? MHC I or MHC II?

Answer 32

- Only present at low level, they present 1-5% of spleen and lymph node T cells - Are predominantly CD4 and CD8 negative - The TCRgamma/delta receptor bind antigens directly, no antigen presentation by MHC I or MHC-II is required - TCRgamma/delta repertoire is very narrow, there are two dominant TCRgamma/delta T cells:

Question 33

Describe the cells that express delta1. What tissues do they populate? What do they recognize?

Answer 33

Cells that express delta1 with various gamma genes: these are the first gamma/delta T cells that emerge from fetal thymus. They eventually populate epithelia tissues such as the intestine and skin. The TCRdelta1 T cells recognize stressed cells and lipid antigens presented by CD1B or CD1C molecules.

Question 34

What are the majority of circulatory gamma/delta T cells?

Answer 34

TCR gamma 9 delta2- 80% of all gamma/delta T cells

Question 35

Describe CD1 proteins.

Answer 35

CD1 proteins transmembrane proteins that are distantly related to MHC molecules; however these proteins lack polymorphism.

Question 36

Describe the four human CD1 proteins.

Answer 36

There are four human CD1 proteins: CD1A, CD1B, CD1C and CD1D, each is encoded by different genes: - CD1B and CD1C are noncovalently associated with B2m (CD1B and C particularly bind to bacterial glycolipid antigens.) - CD1B, CD1C and CD1D are known to bind to glycolipid antigens; CD1B and C particularly bind to bacterial glycolipid antigens.

Question 37

What are the TCR gamma delta functions?

Answer 37

TCRdelta1 cells: lyse stressed/ transformed epithelial cells TCRgamma9delta2: recognize non-peptide pyrophosphomonoester antigens (phosphor antigens) found on mycobacterium and malaria parasite. These cells can also kill intra- and extracellular M. tuberculosis, produce IFNgamma that affects cytotoxicity of NK and NKT cells and the generation of TH1 cells.

Question 38

Describe the development of Natural Killer cells. Where do they develop? From what? Which cells develop into NKT cells? What types?

Answer 38

- NKT cells develop in the thymus from the CD4Pos CD8Pos DP thymocytes. - DP that recognize glycolipid antigens presented by CD1D positive cortical thymocytes develop into NKT cells - NKT are either CD4Pos or CD4NegCD8Neg

Question 39

What do NKT express? What tissues do they populate?

Answer 39

NKT express both the NK maker CD56 and the T cell maker TCRalphaB/CD3 complex - Mature NKT egress the thymus and populate the liver, spleen, BM, and lymph nodes. - Human NKT population constitutes approximately 0.02- 0.2% of the peripheral blood T cell compartment.

Question 40

What are the functions of NKT cells? What happens when they are triggered?

Answer 40

NKT cells rapidly produce both Th1 (IFN-gamma) and Th2 cytokines (IL4, IL5, IL10) upon triggering; thus they play a role in immunoregulation.

Question 41

How were Regulatory T cells (Treg) discovered? Describe the observations that led to their discovery in mice.

Answer 41

- Mice subjected to thymectomy between day 2 and day 4 post natal develop organ-specific autoimmune diseases - The autoimmune diseases can be prevented by infusion of syngeneic T cells obtained from adult thymus or spleen - It was later identified that the CD4Pos CD25Pos T cells are capable of suppressing the autoreactive T cells. From these observations, it was concluded that the thymus generate CD4Pos CD25pos suppressor cells with specificity for autoreactive T cells that may escape negative selection during the development of T cells. This mode of suppression of autoreactivity is referred to as dominant tolerance in contrast with central tolerance in which the autoreactive T cells are deleted.

Question 42

Describe Treg development in humans. Where do they develop (from which cells) and how early?

Answer 42

Develop in the thymus from CD3Pos CD4Pos T cells as early as 14 weeks of gestational age. Treg represent 5-10% of mature CD4pos thymocytes and 10% of peripheral blood CD4PosT cells.

Question 43

Describe the Treg that develop in the periphery. How are they genearated here? What types are there?

Answer 43

Treg can be generated from peripheral mature CD4pos T cells by TGF-B T regulatory type 1 (Tr1) T helper 3 (Th3)

Question 44

Describe Treg and human diseases. What is IPEX? What do patients with IPEX present with clinically? What gene is responsible for IPEX?

Answer 44

Immune dysregulation, polyendocrinopathy, enteropathy, and X-linked inheritance (IPEX) is a clinical syndrome that presents with multisystem autoimmune disease. Clinically, patients with IPEX manifests most commonly with diarrhea, insulin-dependent diabetes mellitus, thyroid disorders, and eczema. FOXP3, the gene responsible for IPEX, maps to chromosome Xp11.23-Xq13.3 and encodes a transcription factor. Because patients with IPEX lack Treg, it was determined that expression of FOXP3 in CD4Pos T cells is required for the development of Treg in the thymus.