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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
lor
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.
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