The B lymphoid lineage 2: subsets

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The B lymphoid lineage 2 subsets

• Simon Hunt
• Dunn School of Pathology
• email simon.hunt_at_path.ox.ac.uk
• You are welcome to use the material in this
  lecture for your own private study further
  distribution is not allowed. Available by link
  from http//www.psb.ox.ac.uk/Newfhs/09imm.htm

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The BIG questions 27 May 2003
Why Humoral as well as Cell-Mediated? How
regulate the choice? Contribution to overall
immunity incl Innate? B cell tolerance How?
Why some AutoAb specificities, not others? IgD
function? Ig expression control, class-switching,
esp to IgE? B memory how? Is there a distinct
memory cell? Affinity Maturn? B cell subsets
- why?? Why doesnt immune system get bigger and
bigger with repeated stimulation?
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Early B cell developmentcells and
genes(Melchers, Rolink, Osmond)
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Early B cell development organisation in
BM(Fleming and Paige)
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Stages in MOUSE BM lymphopoiesis After Hardy and
Hayakawa (2001)
IL7 is not essential in human
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Experimental strategy

• Define specialised cell types
• use mAbs to define CD antigens
• protein product
• example c??(pre-B?, then s??(naive B) IgM as
  antigen receptor
• (in future) differential gene-expression readouts
• Show conversion from precursor to product
• in vitro
• example pre-B to B cells
• in vivo
• Repopulation assays long-term chimaeras

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Complementarity-determining residues in Igs
Wu-Kabat plot
HyperVariable
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Ig Gene Location
?kappa
?lambda
?Heavy
Find your favourite gene via PubMed
http//www.ncbi.nlm.nih.gov/cgi-bin/Entrez/hum_sr
ch?chrhum_chr.infquery
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The germ-line genes for VH regions
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V gene diversification 1

• Rearrangement of DNA of genome
• Example - Heavy chain gene segments

Combinatorial diversity by picking one V, one D,
one J segment from many available
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V gene diversification 2

• Junctional diversity
• Imprecise joins - frameshift or intra-codon
  recombination

• Nucleotide insertion N region diversity
• Enzyme TdT (terminal deoxynucleotidyl
  transferase) nibbles at joins inserts bases - no
  template. DNA repair.

Errors!! Non-productive rearrangements!!
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The numbers how many genes?

• Underestimate because
• No allowance for junctional or N region
  diversification
• Omits occasional D-D joins
• VH and possibly DH show polymorphic alleles, so
  most people are heterozygous

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The germline V genes fall into families

• Sequence analysis shows distinctive family
  resemblances
• Intra-family members more closely related to each
  other than to another family more closely
  related in evolution
• Features based on Framework (i.e. non hyperV)
  parts of the sequences
• Sometimes called clans
• Smart bugs can recognise these features
  superantigens
• E.g. staphylococcal enterotoxin B recognises Vb3,
  7, 8, 17 (of the 20 Vb families in human)
• Staphylococcal Protein A recognises Vh3
• (in addition to well-known Fc-binding activity)

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SpA interaction with Vh3
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Cells in which primary diversification occurs

• Lymphocyte progenitors DH to JH
• Then seeding to Thymus or stay in Bone Marrow

• Thymus
• Try D? to J? then V? to D?
• might make gamma gene
• If not, try other allele
• another go at gamma gene
• Try D? to J? then V? to D?
• etc, etc
• ? Tcr ?? or Tcr ??

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One cell, one specificity

• Clonal Selection Theory needs monospecificity
• Humans are diploid each locus has two alleles,
  from Mum and Dad
• If like all other somatic cells, each B cell
  would make 2 Heavy chain V genes
• There are two Light chain V loci (? and ?)
• Each B cell could make 4 Light chain V genes
• B cell rearrangement stops when one productive H
  and one L is made
• allelic exclusion observe by staining
  heterozygote with two coloured antibodies to each
  Ig allele. Likewise isotype exclusion (? or ?,
  not both)
• feedback system prevents any further
  rearrangement
• Hence Ig transgenic mice are monospecific, since
  endogenous gene rearrangement is blocked
• likewise Tcr genes (but TCR a is exception does
  not respect allelic exclusion)
• Clinical application myeloma diagnosis
• myeloma is a monoclonal tumour (one V region only
  one Idiotype)
• Has unbalanced kappa/lambda ratio

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Normal B cell ontogeny
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Lymphoma cell of origin
From http//www.path.sunysb.edu/hemepath/tutorial/
default.htm
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Lymphochip micro-array of cDNAs from lymphoid
cells
1. Hybridise cDNA from tissue of interest (tagged
one colour) compared with cDNA from reference
tissue (another colour) 2. Measure ratio of the
two fluorescent colours for each spot of cloned
cDNA 3. Computer sorts out high and low binders

17856 cDNA clones
http//www.cdc.gov/ncidod/eid/vol6no5/cummings2G.h
tm
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cDNA gene expression profiling on Lympho-chip
micro-array
13637 Genes heirarchically grouped by computer
after analysis
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cDNA expression profiling on Lympho-chip detail
T lymphocytes B lymphocytes Follicular lymphoma
T
B
F
Purple gt mean Yellow lt mean
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Classification of lymphomas

• http//llmpp.nih.gov/lymphoma/

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Summary

• Progressive development and commitment
• Totipotent stem cells progenitors precursors
  immature mature (naïve)
• Much cellular overproduction (wastage),
  associated with
• Errors in rearrangement -gt non-productive
• Selection against self (see later)
• Pathway is useful in classifying B lineage
  tumours
• Produces a large repertoire for antigen to select
  from
• Some BIG questions left.