Lecture 9- T cell development

•Flow cytometry- how it works•Thymic architecture and cells•Thymic development I- generation of a TCR chain•Thymic development II- vs T cell development•Positive selection of CD4+CD8+ T cells.•Negative selection of high affinity autoreactive cells.•Alloreactivity, recognition of the MHC molecules of others.

Fluorescence

Fluorescence 2

1-10 x 10-9 seconds

Fluorescence 3

Figure 2-13

Fluorescein

Couple to antibody

Green fluorescent protein

Examples of commonly used fluorescent probes

T cell development quality control- phase I-chain selection

• As with B cell development, T cells develop in an ordered sequence.

• First, there is TCR chain gene recombination, starting with D-to-J.

• Next, V-to-DJ rearrangement occurs. As rearrangement is random and error-prone, -chain rearrangement is tested for functional protein using the surrogate chain mechanism (preT).

T cell development occurs in the thymus

Neonatal removal of the thymus, or congenital lack of the thymus(DiGeorge Syndrome, nude mice) leads to T cell deficiency

Scanning electron micrographof thymus

Developing T cell

Thymic stroma

Thymocyte maturation(approximate)

Positiveselection

Negativeselection

V(D)Jrecombination

The first step in T cell development, TCR rearrangementand testing for protein.

Figure 5-6 part 1 of 3

Figure 5-6 part 2 of 3

CD44-25+

Expansion

CD4-8-CD4-8-HSA-

Precursor

CD4-8-HSA+

CD4+8+

CD8

CD4

CD44-25-

lineage

lineage

3-day life

Lineage Commitment

-chain+

• Cells with a good first chain then proliferate a bit, then stop and redirect rearrangements at the second locus, with V-to-J recombination. At this time, preT is no longer expressed.

• In contrast to B cells, T cells that make an intact receptor are screened for "positive selection" based on low affinity for self-MHC/self-peptides.

• Cells that have no affinity for self-MHC/self-peptide complexes are eventually lost by apoptosis.

T cell development quality control- phase II

Thymocyte maturation(approximate)

Positiveselection

Negativeselection

V(D)Jrecombination

The ability of CD8 T cells to recognize MHC class I bound peptides and CD4 T cells to see MHC class II bound peptides

is not random, but is selected for during thymic education.

• Before selection, cells expressing TCR coexpress both CD4 and CD8 and are called "double positive cells".

• These cells, which are the major cell population in the thymus, mature when their TCRs see MHC (carrying self-peptides) with a low affinity.

• The coreceptor (CD8 or CD4) engaged regulates the fate decision to CD8 or CD4 "single positive" differentiation.

• Hence, CD8 cells have TCRs that were selected on class I, and CD4 cells have TCRs that were selected on MHC class II.

T cell populations during development T cell populations during development and in the lymphoid tissueand in the lymphoid tissue

8.3 83.2

4.7

11.4

7.9CD4CD4

CD8CD8

THYMUSTHYMUS SPLEENSPLEEN

Fluorescence color 1

Flu

ores

cenc

e co

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2

Figure 5-5T cells failing positive selection die in the cortex and are immediately engulfed by macrophages. Red stain shows dead cells, blue stain, macrophages.

Thymocyte maturation(approximate)

Positiveselection

Negativeselection

V(D)Jrecombination

T cell development quality control- phase IIINegative selection

•Cells that bind with too high an affinity for self-MHC/self-peptide complexes are lost, probably by apoptosis.

U. of Cambridge

Figure 5-3 part 2 of 2

Figure 5-13

TCR CD8+4+ double positive thymocytes that fail to be positively selected can undergo many rounds of TCR recombination in an attempt to generate a functional receptor.

Figure 5-19

T cell developmental stages are identified by cell surface markers and gene rearrangement status.

A major checkpoint involves the testing of functional TCR for ability to pair with the surrogate TCR chain, preT.

Kuby et al. W. H. Freeman & Co. and Sumanas, Inc.Immunology, January, 1997

Molecular basis of direct allogeneic MHC recognition

Concepts in T cell development

• TCR cells develop in the thymus from bone marrow progenitors.

• TCR is rearranged first and tested in association with pT.

• Cells that express TCR protein then express CD4+CD8 and rearrange TCR genes.

• Cells carrying appropriate TCRs undergo positive selection, leading to expression of just CD4 or CD8.

• Autoreactive T cells undergo negative selection in the medulla.

• The result of selection is a skewing of the T cell repertoire that promotes MHC restricted recognition.

• Thymic selection also leads to skewing toward alloreactivity and high frequency reactivity to allografts.