Immunology Chapter 7

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Chapter 7

Development Of T Lymphocytes

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Chapter 7

The development of T cells in the thymus Stages of gene rearrangement that produce the 1 repertoire of

T-cell receptors Positive and negative selection of T-cell repertoire

Processes of + & - selection that act on the primary repertoire of T-cell receptors in the thymus to produce the circulating population of mature; naïve T cells

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The Development Of T Cells Compared To The Development Of B Cells

Similarities in the development of T and B lymphocytes: Derive from bone marrow stem cells Undergo gene rearrangement antigen receptors

B cells rearrange in the bone marrow Precursors of T cells leave the bone marrow ->thymus

The formation of 2 distinct T cell lineages : Receptors (1-2% of the primary repertoire) : Receptors (not as stringent selection)

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Primary Lymphoid Tissue For T Cell Development Is The Thymus

Major function of the thymus function is to ensure that mature T cells that leave thymus are restricted to the particular MHC class expressed by an individual person (self -MHC)

Two selection processes:Positive selection leads to the death of immature T cells having receptors that do not interact with any self-MHC class I and II

Negative selection induces the death of those immature T cells that are autoreactive (receptors bind too strongly to a self-MHC molecule)

Mature T cell leaving the thymus to circulate in the secondary lymphoid organs is:

1.Rendered tolerant of self-antigens

2.Responsive to foreign antigens

3.Ready to fight infection

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Only 1-2% of T cells exit the thymus and go into circulation.

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The Development Of T Cells In The Thymus

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The Development Of T Cells In The Thymus

T cells originate from bone marrow stem cells Emigrate to mature in the thymus Named thymus-dependent lymphocytes T cells

Majority are : cells Minority are : cells

2 lineages develop in parallel from common precursors While developing in the thymus

T cells also express other cell-surface proteins related to their eventual functions

Examples are CD4 and CD8 glycoproteins

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T Cells Derive From Bone Marrow Stem Cells

From the bone marrow, T cell precursors migrate through the blood to the thymus

Thymus is where the development of T cells occurs

• Mature T cells then leave the thymus in the blood and enter the secondary lymphoid organs, such as the spleen or lymph nodes.

• In the absence of activation mature T cells recirculate between the blood, the secondary lymphoid tissues, the lymph and the GALT.

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T Cells Develop In The Thymus

Thymus is found in the upper anterior thorax above the heart

Immature T cells - called thymocytes - are embedded in epithelial cell network called the thymic stroma

Thymus is primary lymphoid organ: Involved in the development of T cellsNot involved in lymphocyte recirculation via lymphBlood is the only route through which T cells enter

and exit

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T Cells Develop In The Thymus

In the embryonic development of thymus Epithelial cells of cortex outerEpithelial cells of medulla inner Rudimentary thymus called thymic anlage

Is colonized by cells from bone marrow Progenitor cells thymocytes & dendritic

cells populate medulla Bone marrow derived macrophages also

populate medulla (also macrophages scattered throughout the cortex of thymus

Thymocyte mature progressively move from outer subcapsular region to the inner cortex and the medullar

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The Epithelial Cells Of The Thymus Form A Network Surrounding Developing Thymocytes

Scanning electron micrograph of thymus

Developing thymocytes (spherical cells) occupy the interstices of an extensive network of epithelial cells

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Immature thymocytes

Immature thymocytesCortical epithelial cellsMacrophages

Mature thymocytesMedullary epithelial cellsDendritic cellsMacrophages

Mature thymocytes

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The Cellular Organization Of The Thymus

Macrophages in both cortex and medulla remove the many thymocytes that fail to mature properly

Hassall’s corpuscles Characteristic feature of

The medulla (? Sites of cell destruction?)

Thymus= multi-lobal (stained with hematoxylin and eosin, viewed by light microscope)

The darker staining of cortex vs with the lighter stained medulla

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DiGeorge’s Syndrome Example of importance for the development

a functional T-cell repertoire A deletion in chromosome 22 in which the

thymus fails to develop and T cells are absent

Susceptibility to wide range of opportunistic infections resembles SCID (severe combined immunodeficiency disease

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Thymus And Aging

Thymus fully developed before birth; is most active in the young; atrophies with age Progressively shrinks, fat gradually claiming areas once packed with

thymocytes = involution of the thymus Reduced production of new T cells not noticeably impairing T cell immunity

Nor does thymectomy = removal of the thymus affect T cell immunity of adults

Once established, the repertoire of mature peripheral T cells is long lived and/or self-renewing Differs from the mature B-cell = shorted lived cells that are continually being

replenished from the bone marrow

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The Two Lineages Of T Cells Arise From A Common Thymocyte Progenitor

Maturation of thymocytes into mature T cells occurs in distinct steps Marked by changes in the status of the TCR genes Expression of the TCR protein Production of other T-cell surface glycoproteins

CD4, CD8, CD3 complex Changes in cell surface proteins expressed at each developmental

stage is a way to distinguish between subpopulations of developing thymocytes

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The Two Lineages Of T Cells Arise From A Common Thymocyte Progenitor

Progenitor T cells that enter the thymus lack the cell surface glycoproteins (CD4, CD8, CD3) of mature T cells but they do have CD34 (a cell surface glycoprotein of stem cells).

The TCR genes are in germline configuration Upon interaction with thymic stromal cells, the progenitor T cells will proliferate

and differentiate Approximately one week later, progenitor T cells will express the T-cell

specific adhesion molecule CD2 and other surface markers such as CD5 but no TCR complex No CD4 or CD8 called “double negative” thymocytes

IL-7 receptor on T-cells is essential for binding IL-7 secreted by thymic stromal cells – helps tell the T-cell what to do next in its maturation.

Notch 1 – at all stages of maturation in the thymus signals are sent through this receptor to drive the T-cell in their differentiation.

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T-cell Development in the Thymus is Driven by the Receptor Notch 1

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T Cells Have Two Lineages Distinguished By The Expression Of An Or A TCR

Commitment does not occur before TCR rearrangement but is a race to obtain a productive rearrangement

Thymocytes will rearrange their , and -chain genes about the same time Different from B-cell development (recall: each type of Ig gene is

rearranged in turn and in a set order) Productive and -chain gene rearrangement made prior to a

productive -chain rearrangement leads to receptor which signals cell to stop rearrangement of chain

More frequently the chain productively rearranges before the and -chains

-Chain assembles with a surrogate chain = pt (pre-T-cell receptor) which signals the cell to halt rearrangement of , and -chain genes and begin to proliferate

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After expression of the pre-TCR, the recombination machinery is reactivated & targeted towards the chain loci (and the and loci)

In a minority of these cells successful completion of and chain gene rearrangements occurs before the chain gene has rearranged the lineage

In a majority of these cells, productive rearrangement of the -chain gene occurs first an T cell

Recall the chain locus is located within the -chain locus…a rearrangement at an -chain locus results in the deletion of the complete chain locus from the chromosome

T cells have two lineages distinguished by the expression of an or a TCR

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T Cells Have Two Lineages Distinguished By The Expression Of An Or A TCR

Cells committed to one lineage can contain productive rearrangements for the TCR genes of the other lineage (except for -chain).

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Immature T Cells That Undergo Apoptosis Are Ingested By Macrophages In The Thymic Cortex

Failure to make a productive rearrangement results in death by apoptosis (fate of about 98% of thymocytes) Macrophages in thymus continually

remove dead cells Cells have been stained for

apoptosis with a red dye Apoptotic cells are scattered

throughout the cortex but are rare in the medulla

Higher magnification red for apoptotic cells and blue for macrophages

Apoptotic cells are visible within macrophages

cortex

medulla

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Production of T-cell receptor β chain stops β-chain rearrangement and leads to expression of CD4 and CD8

Receptor most abundant type found on T cells.

TCR -chain locus = variable (V), diversity (D) and joining (J) gene segments and is rearranged first (similar to heavy chain in Ig’s)

TCR -chain locus has no D segments and is rearranged after the -chain (similar to light chain in Ig’s)

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Production of T-cell receptor β chain stops β-chain rearrangement and leads to expression of CD4 and CD8

Production of a functional -chain gene -chain translation and assembly with a surrogate -chain (preT), CD3 proteins and chain to form a pre-T cell receptor transported to the cell surface

Role of pre –T-cell receptor is analogous to the pre-B-cell receptor in B-cell development Triggers the thymocyte to

proliferate and halt -chain gene rearrangement

Ensures only one type of T-cell receptor -chain is expressed by the T cell

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Unproductive Rearrangement At One -Chain Locus Can Lead To Rearrangement Of The -Chain On The Homologous

Chromosome, Rearrangement At the Same Locus Can Also Occur

This is not the case for Ig H-chain genes Exists with T-cell -chain because 2 sets of DJ and C gene

segments are tandemly associated with the V gene segments.

Potential to “try out” up to 4 gene rearrangements = 80% of T cells make successful rearrangement of the -chain gene.

Only 55% success rate for productive H-chain gene rearrangement by B cells.

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Rescue Of Unproductive -Chain Gene Rearrangements

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Unproductive Rearrangement At One -Chain Locus Can Lead To Rearrangement Of The -Chain On The Homologous

Chromosome, Rearrangement At the Same Locus Can Also Occur

Successful rearrangement of a -chain gene induces expression of the two co-receptors (CD4 & CD8) Called “double-positive” thymocytes Found predominantly in the inner cortex of the thymus Interact intimately with the network of epithelial cells

During cell proliferation initiated by signaling through the pre-TCR, expression of the RAG-1 and RAG-2 genes is repressed (allelic exclusion of the beta chain)

No rearrangement of the -chain genes occurs until the double-positive cells stop dividing Ensures each cell with a productive -chain gene rearrangement

produces many daughter cells that have the potential to express a different -chain gene

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T-cell Receptor -Chain Genes Can Undergo Several Successive Rearrangements

The TCR -chain can undergo several successive gene rearrangements

Presence of many V and over 50 J gene segments allows many successive rearrangements (like Ig )

Almost every developing T cell will make a productive -chain rearrangement

The chain loci is much larger than the -chain loci which adds to is flexibility

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Successive Gene Rearrangements Allow The Replacement Of One T-cell Receptor Chain By Another

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Checkpoints in T-cell Development

Check-point 1 - Once a beta chain is produced it is sent the ER to make sure it can bind to the surrogate alpha chain (pT) – Pre-TCR

Check-point 2 – Once an alpha chain is made it is sent to the ER to make sure it can bind to the beta chain - TCR

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Stages of T cell development correlate with TCR gene rearrangement and expression of cell surface proteins.

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Positive And Negative Selection Of The T-cell Repertoire

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Positive And Negative Selection Of The T-cell Repertoire

• Second phase of T cell development involves selection of T cells bearing TCRs that can recognize an individual’s own MHC presenting peptides.

• This selection process involves only T cells and not T cells.

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T-cells That Can Recognize self-MHC Are Positively Selected In The Cortex of the Thymus

• Gene rearrangement produces a repertoire of T cells bearing TCRs that can interact with the hundreds of MHC class I and II molecules present in a population.

• Therefore, TCRs expressed on the T cells of one individual are not made to specifically interact with only that individual's MHC molecules.

• Only a small population (2%) of double positive thymocytes will be able to bind to a specific MHC the rest die by apoptosis in the cortex.

• Positive selection is the process by which this small population of T cells that reacts with the individual’s own MHC molecules is selected.

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T-cells That Can Recognize self-MHC Are Positively Selected In The Thymus

• Positive selection takes place in the cortex of the thymus and is mediated by cortical epithelial cells bearing complexes of class I and class II self-MHC and self-peptides.

• Cortical epithelial cells form web of cell processes that envelope CD4,CD8 double-positive thymocytes.

• At the point of contact – interactions between the TCR of thymocytes with self-MHC and self-peptide are tested. If a peptide:MHC complex is bound by a thymocyte within 3-4

days of expressing a functional TCR, then a positive signal is delivered to the thymocyte.

A thymocyte that does not receive a signal dies by apoptosis and is removed by macrophages.

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T-cells that can recognize self-MHC are positively selected in the thymus

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T-cells that can recognize self-MHC are positively selected in the thymus

• Self-peptides presented in the MHC molecules of cortical epithelial cells are derived from self-proteins present in the thymus.

• The number of different peptides that can be presented by one individual’s MHC molecule is estimated to be about 10,000.

• For someone who is heterozygous for the six major HLA genes about 120,000 self-peptides could be presented by 12 different MHC class I and II molecules.

• The mature T-cell repertoire is about tens of millions or more, so most of the self-peptides:MHC complex will bind a T cell receptor during positive selection.

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Positive Selection Controls Expression Of The CD4 Or CD8 Co-receptor

• Positive selection also determines whether a double positive thymocyte matures into a CD8 or CD4 T cell, known as “single-positive” thymocytes.

• CD4 T cells only interact with MHC class II molecules and CD8 T cells interact with MHC class I molecules.

• During positive selection…When a CD4 CD8 double-positive thymocyte interacts

through its TCR with a class I MHC molecule, CD8 is recruited and CD4 is excluded.

When a CD4 CD8 double-positive thymocyte interacts through its TCR with a class II MHC molecule, CD4 is recruited and CD8 is excluded.

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Rearrangement Of -Chain Stops Genes Once A Cell Has Been Positively Selected

• Rearrangement of the -chain locus continues throughout the 3-4 days of positive selection – hence a T cell can change the specificity of the TCR it expresses.

• Once a T cell is positively selected, rearrangement of the -chain stops.

• Some double positive thymocytes can express two -chains (one from maternal allele and one from the paternal allele) and thus two types of TCR and undergo positive selection by engagement of one of these receptors.

• The number of positively selected cells is very small, therefore it is rare that one cell will have two selected TCRs (one receptor is usually nonfunctional).

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T-cells Specific For Self-antigens Are Removed In The Thymus By Negative Selection

• Negative selection serves to delete T cells whose antigen receptors bind too strongly to the complexes of self-peptides and self-MHC molecules presented by thymic cells.

• Negative selection is mediated by several cell types, most important of which are the bone marrow-derived dendritic cells and macrophages.

• Engagement of the MHC molecule of one of these specialized thymic antigen-presenting cells by the TCR of a thymocyte causes that cell to undergo apoptosis and phagocytosis by macrophages.

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• Negative selection cannot eliminate T cells bearing TCRs that can bind to self-peptides not present in the thymus – such cells enter the periphery but are rendered anergic or inactivated.

• How the positive and negative selection processes used in the thymus lead to death or growth is not well understood but likely involves differences in the affinity of the ligand-receptor interactions.

• Because of the diversity of HLA types in a population an individual’s T cell component becomes highly personalized.

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Regulatory T cells

CD4 T-cells Express

CD25 FoxP3 a transcriptional repressor is used by the regulatory T-cells

(unique to regulatory T-cells)

Distinct from naïve T-cells Contact with MHC II – self-antigen can suppress proliferation

of naïve T-cells responding to self-antigens Suppressive effects require contact between the two T-cells

and secretion of non-inflammatory cytokines

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T-cells Undergo Further Differentiation In Secondary Lymphoid Tissues After Encounter With Antigen

• The T cells that survive the selection processes in the thymus become mature, naïve T cells that recirculate through blood into the secondary lymphoid organs.

• Mature T cells are longer-lived than B cells and in the absence of specific antigen stimulation will continue to circulate in the body for many years.

• The T cell zones of lymphoid organs are sites where naïve T cells are activated by antigen – which provokes the final phase of T-cell development and differentiation.

• Mature T cells become effector cells that can stay in lymphoid tissues or migrate to sites of infection.

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T-cells undergo further differentiation in secondary lymphoid tissues after encounter with antigen

• There are several different types of effector T cells.• CD8 T cells become activated cytotoxic T cells.• CD4 T cells differentiate under the influence of cytokines

into TH1 or TH2 helper T cells.• Which type of CD4 T cell predominates depends on the

nature of the pathogen and immune response needed.• In an healthy individual there are about twice the number

of CD4 T cells to CD8 T cells.• In patients with AIDS this proportion changes becomes

the AIDS virus infects and kills CD4 T cells.

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Stages of T-cell Development in the Thymus

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Stages of T-cell Development in the Thymus

Immunology Chapter 7

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