CMB2004 lecture 4

Lymphocyte development

Bone marrow to specialised immune cells

B cell development​

• Develop from haematopoietic stem cells in bone marrow that express PAX5 transcription factor ​

• Continuous process: ~3 x 1010 produced per day​

• Involves re-arrangement and expression of Ig genes​

• Expression of lymphocyte, and then B cell-specific, markers e.g. CD45, then CD19​

• Removal of self-reactive cells (negative selection in attempt to avoid autoimmunity)​

precursor B cell to plasma cells

pre-B cells​

1. H chain genes rearrange first (m chain) ​- moves to cell surface with Ig alpha and Ig beta and expressed with surrogate light chain (product of V preB and l5 genes) ---> pre-B cell receptor (pBCR)

then light chains rearrange, and displace ​V preB and l5 chains (associating with H chain) ​--->IgM BCR

immature B cells (still in BM)​​​​

Formation of pre-B cell receptor​

Rearrangement of Ig genes during B cell development​

pre-BCR​

• Delivers signal to pre-B cell that H chain looks functional​

• no Ag required yet​

• ​

• Signal from pre-BCR:​

• Turns off RAG-1, RAG-2 genes​

• 5 - 6 rounds of cell division​

• Surrogate light chain expression stops​

• RAG-1 and RAG-2 turned on again​

• L chain rearrangement starts​

• (RAG genes needed for gene rearrangement)​

Light chain possibilities:

each individual B cell expresses either a kappa (k) or lambda (l) light chain​

As k light chain genes rearrange before l genes, ​more B cells will express k than l light chains​

Immunoglobulin gene re-arrangements​

• error prone​

• if cell fails to productively re-arrange both H and L genes,​ it dies​

• following successful H chain re-arrangement, pre-B cells ​that initially fail to generate non-productive re-arrangements of ​light chain k genes can be “rescued” by up to 10 further ​rearrangements at the same locus, (as there are 5 Jk genes on​ each chromosome). If after all these attempts and still out of frame, ​then l locus will begin to rearrange​

repeated rearrangements at light chain

Proteins expressed during B cell development​

Tolerance in immature B cells​

• Express membrane IgM only​

Immature B cells that:​

• bind multivalent self-antigen undergo either​

• clonal deletion: cell dies by apoptosis​

or​

• receptor editing: further light chain gene rearrangements of variable regions (get another chance)​

• ​

• bind soluble self-antigen--> cell becomes unresponsive​ (anergic)​

​

what happens when immature B cells have self antigen binding?

death or inactivation​

Qs

• What is the origin of haematopoietic stem cells?​

• Site of B cell development to the stage f immature B cell?​

• Most predominant sIg (BCR expressed by immature B cells?​

• If BCR expressed by immature B cell binds to self cell surface antigen, the B cell is removed by which type of selection?​

• Activated B cells in the periphery can develop into?​

• Which Ig chain is re-arranged first?​

• What is the surrogate light chain?​

• Which gene segments get re-arranged in the heavy and light chains?​

• Which stage of B cell development sees the most cell division?​

• What do the RAG1 and 2 genes do and when?​

• What is the fate of a pre-B cell with non productive re-arrangements of light chain genes?​

• If the BCR expressed on an immature B cell binds multivalent self-antigen what are the 2 possible outcomes for that B cell?​

T cell lymphocyte development

T cell development​

​

precursor to active T cells

a/b T cell development​

Precursors produced in bone marrow, migrate to thymus​

once in the thymus develop into thymocytes:​

• ​

1) re-arrange TCR genes (β first) and express TCR​

2) acquire other markers e.g. CD3, CD4, CD8 (all 3 to start)​

3) undergo positive and negative selection (thymus)​

The thymus​

• bi-lobed organ in anterior mediastinum​

• each lobe divided into many lobules​

• each lobule has outer cortex and inner medulla​

cells: lymphoid cells​, epithelial cells​, macrophages and dendritic cells (APCs)​

T cell maturation in thymus​

• pro-thymocytes enter cortex via blood vessels from bone marrow​

• ​

• once inside thymus, thymocytes then re-arrange TCR genes​

• firstly, they rearrange TCRb genes (similar to H chain in BCR)​

• expressed along with pre-T cell receptor​

• cells proliferate and then re-arrange TCRa genes​

• express TCR together with CD3​

• also express both CD4 and CD8 ​

​

• (note: peripheral T cells express one or the other)​

• ​

TCR complex​

gamma delta expression in thermocytes

• similar structure to ab receptor (different loci)​

• gd T cells do not express CD4 or CD8 markers​

• ​

• less diversity than αβ receptor​

• expressed on separate T cell population​

• 1-5% in circulation​

• majority in epithelial tissues at mucosal surfaces​

• ​

• recognise different antigens from ab T cells​

• ​

• lineage commitment to gd or ab depends

thymocyte production

T cells expressing a randomly rearranged a/bTCRs may:​

1) recognise self MHC plus peptide from “foreign” Ag(immunity)​

2) recognise self MHC plus peptide from ”self” Ag(autoimmunity)​

3) not be able to recognize self-MHC ​

(as all cells will express self-MHC, T cells with this TCR will be useless)​

​

need to keep T cells with TCR 1, eliminate those with TCR2 and 3: positive and negative selection. ​

reality is a fine balance based on TCR/MHC affinity​

how is selection performed?

spatially and timely ​

1. Positive selection of T cells:​

• positive selection of cells which recognise self MHC (+ selfpeptide)​

• occurs when double-positive (DP, CD4+CD8+) T cells recognise MHC on cortical epithelial cells in thymus​

• -> apoptosis if not recognised​

• rearrangement gives random TCR repertoire​

• only 1 - 2% capable of recognising own MHC​

• cells expressing TCR that doesn’t bind self MHC die via apoptosis​

• positively selected cells move to medulla ​

Negative selection of T cells​

• negative selection of T cells which recognise self MHC (+selfpeptide) on thymic dendritic cells/macrophages with high affinity (as these T cells may induce autoimmunity if not removed)​

• TCR binding to MHC/self-peptide with high affinity causes ​

T cell to die apoptosis (clonal deletion)​

positive and negative selection link

occur sequentially in diff thymus regions

Paradox of positive and negative selection​

• T cells positively and negatively selected on self MHC+ self peptide​

• ​

• Why aren’t all T cells that have been positively selected subsequently eliminated by negative selection?​

TCR affinity for p/MHC is basis for selection:​

all T cells recognising self MHC/self peptide are positively selected​

those with the highest affinity TCR are then negatively selected​

ULTIMATE GOAL​

a population of T cells with low affinity for self peptide + self MHC

These cells represent the safest/most useful to keep with the highest probability they will have a high affinity for self MHC when presenting peptides derived from pathogens​​

abc affinity?

Thymocyte development: summary​

T cells that survive thymic selection (<5%)​

• express TCR capable of binding self MHC​

• are depleted of self-reactive cells​

exit the thymus as mature, single positive (SP) T cells​

​CD8+ T cells recognise Ag in association with MHC class I ​

​CD4+ T cells recognise Ag in association with MHC class II ​

Qs

• Origin of thymocytes?​

• Process of elimination of self-reactive T cells?​

• Site of T cell development and body location?​

• Gene segments for alpha and beta chains that get re-arranged?​

• Cells other than thymocytes present in the thymus are?​

• Alpha or beta genes rearranged first?​

• What is the pre-TCR?​

• What does the CD3 molecule do and why is it important?​

• Do gamma delta T cells normally express CD4/CD8?​

• What is positive and negative selection of T cells?​

• If a T cell recognises peptide and MHC I which markerdoes it have on the surface?​

• Are the criteria for positive and negative selection ofimmature T cells the same?​