B- and T-cell Ontogeny

• Describe the B cell developmental stages
  • Haematopoietic or Pluripotent stem cell (HSC):
    • Can divide into any lineage
  • Common lymphoid progenitor cell:
    • Multipotent, can differentiate into B or T-cell ****
  • B progenitor cell (pre-pro-B cell):
    • ****Earliest recognizable stage, no heavy chain, no light chain
  • Pro-B cell:
    • D to J rearrangement (early) and V to DJ rearrangement (Late) of the IgH chain gene via RAG1 and RAG2
    • Germline IgL chain gene
    • No Ig protein
    • IL-3, IL-7, IGF-1, SCF are required for growth
    • Express: Pax-5, CD19, HLA-DR, CD34
  • Large Pre-B cell:
    • V to DJ rearrangement of IgH chain gene
    • Germline IgL chain gene
    • pre-BCR
    • Stage implicated in X-linked (Bruton) agammaglobuliemia
  • Small Pre-Bcell:
    • VDJ rearranged IgH chain gene, ****
    • V to J rearrangement of IgL chain gene,
    • Intracellular $\mu$ receptor
    • Express: CD79a, CD10, CD20, CD22, TdT
  • Immature B cell:
    • VDJ rearranged IgH chain gene
    • VJ rearranged IgL chain gene
    • IgM protein on the cell surface
    • Locate in the Bone Marrow (BM)
    • Negative selection occurs at this stage
    • Express: HLA-DR, CD19, CD20, CD40
    • Stops expressing CD10, CD34, RAG1, RAG2, TdT
  • Mature naive B cell:
    • VDJ rearranged IgH chain gene
    • VJ rearranged IgL chain gene
    • IgM and IgD on the cell surface
    • Located in the spleen and secondary lymphatic organs
    • Express: surface IgM, IgD, CD19, CD20
  • Antigen-activated B cell (Plasma cells and memory B cells):
    • ****Undergoes somatic hypermutation and class switching after antigen exposure
    • Long-lived cells from lymph follicles are found in the BM
    • Short lived cells are non-follicular
    • Express: Bcl-6, Pax, CD19, CD20
    • Described as: CD38+, CD138+
• Briefly describe Positive and Negative selection as it occurs in T-cells
  • T-cell precursor: Committed to T-cell lineage, Migrates to the thymus, does not express surface proteins
  • Double negative stage (Pre-TCR): Somatic recombination of Beta TCR chain, proliferate, express pre-TCR on surface, express CD3
  • Double positive stage: Somatic recombination of alpha TCR chain, Positive selection at cortex, express CD4, CD8, TCR
  • Single positive: Migrate to the medulla of thymus, Negative selection against self antigen,
  • Mature T-cell: Exit into blood stream and migrate btw peripheral tissue, blood vessels and secondary lymphoid organs, to await peripheral activation, Express TCR, either CD4 or CD8
• Describe negative selection as it occurs in B cells
  • Immature B cell stage
    • IgH chain genes have already undergone VDJ recombination and the IgL gene has undergone VJ recombination
  • IgM is expressed on the cell surface
  • If IgM interacts strongly with membrane bound self-antigens (such as those bound on MHC), the B-cell undergoes:
    • Apoptosis OR,
    • Receptor editing
  • This prevents the formation of self-reacting B-cells
• Briefly describe somatic hypermutation and antibody class switching
  • Somatic hypermutation
    • Occurs during subsequent antigen exposures to contribute towards affinity maturation, Point mutations that create random alterations in the variable region of the antibody gene, mediated by activation-induced cytosine deaminase (AID) and uracil nucleoside glycosylase (UNG), allows error prone DNA polymerase to introduce mutations
  • Isotype (class) switching
    • Activated B cells within germinal centres change the antibody isotype in response to cytokines secreted by T-cells, antibody gets different function but same affinity,
    • Mediated by switch recombinase
    • First signal is by Antigen presentation by MHCII to TCR of Th cell, Secondary costimulatory signal by CD40 and CD40L,
      • IL-4 and IL-13 for IgE,
      • IL-5, TGFB for IgA,
      • IL-6 for IgM,
      • IFNy, IL-4, IL-21 for IgG
• Outline the clinical applications of B and T cell ontogeny
  • Determines antibody response to antigens (T dependent and T-independent response)
  • Structural organization of Lymph nodes (Follicles + Germinal centres have B-cells, Cortex mainly composed of T-cell)
  • Central tolerance in B and T cells (negative selection)
  • Congenital immunodeficiencies involving T and B cells (Bruton’s agammaglobulinemia, DiGeorge syndrome)
  • WHO classification of haematological malignancies (AML, ALL, CML, CLL)
  • Diagnosis of lymphomas by flow cytometry (B-cell CD10, CD19, CD20; T-cell CD3, CD5, CD7)

Differentiate between somatic recombination and somatic hypermutation

• Briefly describe the subsequent stages that follow after immature B cells leave the bone marrow up to antibody generation (write B cell activation in detail)
  • Mature naive B cell:
    • VDJ rearranged IgH chain gene, VJ rearranged IgL chain gene, IgM and IgD on surface, Located in the spleen and secondary lymphatic organs, Express surface IgM, IgD, CD19, CD20
  • B cell activation
    • Exposure to antigen or polyclonal mitogens causes activation and proliferation
    • T dependent or T-independent activation causes the B-cell to undergo Somatic hypermutaion, Class switching and Affinity maturation
    • Memory B cell and Plasma cells are formed
    • Interaction (In-detail)
  • Plasma cell
    • Secrete antibodies
    • Long lived from follicles
    • short lived cells are non-follicular,
    • Express: Bcl-6, Pax-5, CD19, CD20 positive
    • Described as CD39, CD138 positive
  • Memory B cell
    • Identical to resting mature B cell
    • More sensitive to antigen stimulation,
    • Produce high affinity antibodies and persist for many years