THE LYMPHOID SYSTEM I

B- CELL AND T-CELL DEVELPMENT

Haematopoiesis

B-CELL DEVELOPMENT

Introduction

• B-cells function in antibody production against target antigens, present antigens to T-cells and provides signal for T-cell activation.

• Development starts in the fetal liver, and bone marrow (BM) from 12-16 week GA.

• B-cells are identified by surface protein structures—BCR, membrane bound or surface Ig

• Maturation is supported by BM stromal cells

Bone marrow

• Progenitor B-cells migrate from the periosteal region to the centre of the BM, acquire markers of differentiation and maturation. 1 = 64 in 3-4/7. Ig gene rearrangement starts here.

• Pre-B-cells; 1st recognizable stage, appearance of cytoplasmic H chain of IgM, cells do not have fully arranged antigen receptor.

• Immature B-cells; H chain associates with L chain. Positive and negative selection takes place, but cells are not ready to respond to antigens

Spleen and lymph nodes

• Virgin B-cell has fully rearranged Ig genes but have not encountered antigen, expresses IgM and IgD.

• Mature B-cells have encountered antigen and possess antigen specificity, somatic hypermutation and class switching occurs with Ag encounter.

• A) Memory B-cells maintain memory of antigen encounter and reside in the lymphoid system. May die after several years if a secondary response does not occur.

• B) Plasma cells have lost all SIg but remain committed to the production of a single Ab specificity with a single L and H chain type.

Colony stimulating factors

• Stem cell factor (SCF); membrane bound cytokine present on stromal cells, stimulates growth of HSCs and earliest B-cells. It interacts with Kit on precursor cells.

• IL7 is secreted by stromal cells and is essential for growth and survival of developing B-cells

• Thymic stroma-derived lymphopoietin (TSLP) promotes B-cell development in embryonic liver.

Adhesion molecules and chemokines

• VLA-4 and VCAM • Lymphoid progenitor cells and early pro-B cells bind to the

adhesion molecule VCAM-1 on stromal cells through the integrin VLA-4 and also interact through other cell-adhesion molecules (CAMs).

• These adhesive interactions promote the binding of the receptor tyrosine kinase Kit on the surface of the pro-B cell to stem-cell factor (SCF) on the stromal cell, which activates the kinase and induces the proliferation of the B-cell progenitors

• CXCL12 aka stromal cell derived factor helps retain developing cells within the BM.

• Stromal derived factor 1 (SDF)

Transcription factors

• E2A

• Early B-cell factor (EBF)

• PAX-5

• B-cell activation protein (BASP)

T-CELL DEVELOPMENT

Introduction

• Developing T-cell precursors migrate to the thymus to mature, embedded in the thymic stroma.

• Progenitor cells lack surface markers characteristic of mature T-cells and their receptors are un-rearranged.

• Produces 2 distinct lineages ;γ/δ and α/β. • Notch 1 signal switches on specific genes and

instructs the precursor to commit to T-lineage and choices such as α/β vs γ/δ and CD4 or CD8

Overview of T-cell development

Double negative (DN) thymocytes

• Differentiation is followed by proliferation and expression of the 1st surface marker specific for T-cells---CD2+ (Double negative stage, i.e CD4- , CD8-). DN stage divided into 4;

• DN1-- expresses kit, CD44 (adhesion molecule) • DN2—kit, CD44+, CD25+ (α-chain of IL2) • DN3—kit, CD44 ↓, TCR gene rearrangement

occurs and cells that successfully rearrange lose CD25 and move to

• DN4– stage of proliferation.

Double positive thymocytes

• Occur deeper in the cortex

• Thymocytes with CD3 molecules (β-chain expression), expression of CD4 and CD8

• Positive and negative selection occurs here

• Negative selection removes thymocytes with specificity for self-antigen.

• Positive selection rescues thymocytes with specificity for self-MHC.

Single positive thymocyte

• DP thymocytes lose expression of either of CD4 or CD8

• Negative selection ( Reaction to self antigen)

Positive and negative selection

• Estimated time from entry of T-cell progenitor into the thymus and exit of mature T-cells is 3weeks (in mice)

• Thymocytes at different developmental stages found in distinct parts of the thymus

• Cortex; most T-cell developmentt takes place here

• Medulla; Only mature SP thymocytes are seen here. Negative selection takes place here.

Developmental stages at distinct parts of the thymus

Important proteins in thymocyte development.

• Lck– tyrosine kinase

• Terminal deoxynucleotidyl transferase(Tdt)

• ZAP-70; expressed from DN stage, promotes development of SP from DP thymocytes.

• Fyn; expressed from DP stage, essential for devt of α/β thymocytes.

• RAG1/RAG2

Transcription factors

• Ikaros and GATA3 ; expressed in early T-cell progenitors, absence of either disrupts T-cell devt.

• Ets-1;

• T-cell factor 1( TCF1); Expressed in DN stage

Migration of thymocytes controlled by chemokines

IMMUNOGLOBIN /TCR GENE REARRANGEMENT

• Ig/TCR chains are coded for by a gene complex with segments within this complex that code for the variable( V) and constant (C) regions.

• For the L chain, the gene complex is on chromosome 22 for IGƛ, and chromosome 2 for IGĸ.

• The gene segments are the V, J and C segments coding for the variable, joining and constant regions respectively.

• The H chain genes are on chromosome 14, and the gene segments are V, J, an additional segment - the D segments, and the C segment.

• These variable region gene segments have multiple copies; 40 V-segments and 5 J-segments for IGĸ, 30 V-segments and 4 J -segments for IGƛ. The heavy chain gene has 40 V-segments, 25 D-segments and 6 J-segments.

Ig gene segments

• Occurs in the bone marrow. • V gene and J gene segments join together creating an exon

that will code for the variable region of the light chain. • This re-arranged gene is then brought close to the C-gene

segment but separated by an intron; the intron is then spliced resulting in the joining of the C-gene segment with the VJ gene segments to form the complete gene segments that would code for the IG L-chain.

• A similar process occurs for the H- chain, but in this case, there are 3 gene segments. The DH joins with JH segments first, these are later joined by the V-gene segment to form the complete gene segments that would code for the V-region of the H-chain.

• They are then re-arranged to the C-gene segment to form the complete gene segments that would code for the complete H-chain.

• Since there are multiple copies of these gene segments, random selection and re-arrangement of any type results in the different V-regions and thus, diverse antigen specificities --- combinatorial diversity

• RAG1and RAG2 cuts the phosphodiester bonds in the DNA creating a hairpin appearance, Ku, Artemis and DNA ligase IV involved in the joining and repair of the DNA.

• Immunoglobulin gene re-arrangement also involves junctional diversity where point mutations are introduced at junctions between gene segments by the addition and deletion of P and N- nucleotides.

• This process is catalysed by the enzyme, terminal deoxynucleotidyl transferase (TdT).

• It increases the diversity of antibodies produced by 3 × 10 6

• TCR variable region genes rearrange in a manner similar to Ig genes

• α-and γ- chain(chromosome 14 and 7) like Ig L chain is encoded by V,J and C gene segments

• Β- and δ-chain (chromosome7 and 14) like IgH chain by V,J,C and D gene segments

• Rearrangement of the TCR α and β chain gene segments results in VJ and VDJ respectively.