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3. B Cell Development
M1 – Immunology Sequence
Winter 2009

4. 1. Receptors and cells in innate immunity.
2. The two fundamental stages of B cell
differentiation.
3. The steps in antigen-independent B cell
differentiation.
4. How is possible for a human to express
more than ten million antibodies?
5. How are the genes for antibody variable
regions organized?

5. Innate immunity is that protection against
pathogens which is rapid and does not
require specific recognition of the pathogen.
Toll-like receptors (TLRs) are used to
recognize bacteria and viruses in innate
immunity. They are named after similar
receptors in Drosophilia. One function of
these receptors in Drosophilia is to induce
immunity against fungi.

6. Receptor Ligand(s)
TLR1 triacyl lipoprotein
TLR2 lipoproteins, peptidoglycan,
lipoteichoic acids
TLR3 double-stranded RNA
TLR4 lipopolysaccharide
TLR5 flagellin
TLR6 diacyl lipoproteins
TLR7, TLR8 single-stranded RNA
TLR9 unmethylated CpG DNA
TLR 11 profilin
TLR12, TLR13 unknown

7. TLR recognition displays only limited
specificity. TLRs bind to pattern molecules
that are not expressed by humans, but are
shared by groups of pathogens.
TLRs are expressed by many cells, including
leukocytes.

8. Binding to pattern molecules results in signal
transduction from the TLR to the nucleus.
Consequences of TLR signaling:
• Production of cytokines and chemokines
and subsequent inflammation.
• Production of Type I interferons (α and β)
• Killing or inhibition of viruses and
intracellular bacteria.
• Upregulation of co-stimulatory molecules
that help to activate T and B lymphocytes.

9. NOD receptors are another set of receptors
associated with innate immunity. They are
expressed in the cytoplasm.
NOD: containing a nucleotide-binding/
oligomerization domain

10. Several cells are associated with innate immunity:
• Macrophages and neutrophils
• B1 B cells—make most of the antibody in serum.
These antibodies tend to bind common epitopes on
pathogens.
• Dendritic cells, γδ T cells, NK T cells (Dr. Chang)
The innate B and T cells tend to have a set of
antigen receptors with a limited diversity.

11. NK cells are lymphocytes that may be a little larger
and more granular than T and B lymphocytes. They
kill cells that do not express MHC class I molecules
(for example, those infected with viruses or tumor
cells). Their killing is regulated by a complex
interaction among several inhibitory (for example,
binding to some class I MHC inhibits killing) and
activating receptors.
Some NK cells have an Fc receptor that allows them
to bind antibody that is bound to a cell, and kill that
cell by a process called antibody-dependent, cell-
mediated cytotoxicity (ADCC).

12. Immature
Mature
Stem cell
Pro B cell
Pre B cell
B cell
B cell
Y
Y
Y
µ
IgD and IgM
µ chain in
IgM on
on cell
Absent
Absent
cytoplasm
cell surface
surface
University of Michigan Department of Microbiology and Immunology

13. B cell differentiation occurs in the bone marrow
from pluripotent stem cells.
Pro B cells can be distinguished from stem cells
by the expression of several CD antigens: CD19,
CD20, etc.
B cell development depends on the adherent cell
part of bone marrow (stromal cells) and
cytokines (IL-7).
The final product is a mature B cell, which has
never been exposed to antigen (it is naïve).

14. CD molecules (cluster of differentiation).
These molecules are found on the surface of a
group of cells that are at the same state of
differentiation. The group of cells can be large
(all lymphocytes) or small (CD8+ T cells).
Thus, the expression of a CD molecule can be
used to define the state of development of a
cell. They are detected by monoclonal
antibodies.

15. Flow Cytometry (FACS).
This instrument is used to detect the cell
surface expression of CD molecules.
Bind an antibody that is tagged with a
fluorescent molecule (fluorescein,
phycoerythrin, or rhodamine, for example) to a
cell surface molecule.
Analyze amount of antibody bound, and
therefore expression of the cell surface
molecule, in the flow cytometer.

16. Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997

17. If one uses two different fluorescent tags on two
different antibodies, excites them with two
different lasers, and detects them at two
different emission wavelengths, then one can
monitor two cell surface molecules at once. (Or
even more.)
Data presentation: Each cell is represented as
one dot. For single colors, data are often
presented as the number of cells versus
relative fluorescence.

18. Normal
Immunodeficient Immunodeficient
Minegishi et al., J. Clin Invest. 104: 1115-21 (1999)
Pro and Pre
B cells
B cells
Stem cells

19. Antigen-independent B cell differentiation
from a pre B cell to an immature B cell
depends on positive signaling through a
receptor with the mu heavy chain only
(the pre B cell receptor).

20. Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997

21. Bruton s agammaglobulinemia
• Boys with repeated infections by
encapsulated bacteria or sometimes viruses
• X-linked
• Failure to produce antibodies
• Almost no production of immature B cells in the
bone marrow
Mutation in Bruton s tyrosine kinase (btk) that is
activated upon engagement of the pre B cell
receptor. A signal from mu on the surface of pre
B cells in these boys does not reach the nucleus
—positive signaling fails.

22. Antigen-independent B cell differentiation
in the bone marrow results in ten million
different clones of B cells, each with an
antibody on their surface that binds a
different epitope. (The sequences of
heavy chain variable region and the light
chain variable region are different for
every B cell clone.) Thus, the receptor
repertoire is ten million.
(A human has more than 1012 B cells).

23. Each antibody is made by one clone of B
cells. Hence, antibodies are clonally
distributed .
Regents of the University of Michigan

24. Immature B cells with an
immunoglobulin on their surface
that binds to a self antigen sends a
negative signal, resulting in deletion of
the immature B cells in the bone
marrow.
Clonal deletion
This is part of self-tolerance
for B cells

25. Regents of the University of Michigan

26. Allelic exclusion: In a B cell clone, only one of the two
antibody loci (one of the two homologous
chromosomes) is expressed as antibody protein.
Therefore, even though a B cell could express two mu
heavy chains, it only expresses one. Even though a B
cell could express four light chains, it expresses only
one.

27. B cell diversity of more than ten million clones is
generated during antigen-independent B cell
development. How is this diversity generated?
A hint comes from the fact that antibodies are two-part
proteins.
Constant region--one gene. If there were two genes,
single amino acid changes would occur and there
would soon be two isotypes: λI, λII, λIII, etc.
Variable region--many genes for the three kinds of
variable regions: Vκ, Vλ, and VH.

28. Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997
Vκ encodes amino acids 1-95
Jκ encodes amino acids 96-107
Cκ encodes amino acids 108-214

29. Human Vκ
1500 kb of DNA. V genes are 5 to 20 kb
apart.
Most 3 Vκ is 23 kb 5 of Cκ.
Five Jκ regions, encoding amino acids 96
to 107, lie 1.2 kb 5 of Cκ.

30. Of the 79 Vκ genes, almost one-half are
pseudogenes--genes that cannot be
expressed as a light chain, because they
have an in frame stop codon, lack an
invariant amino acid, or are truncated at
the 5 end.

31. V codon 95 CACAGTG--spacer--GGTTTTTGT
ACAAAAACC--spacer--CACTGTG J codon 96
Each V is followed by CACAGTG--spacer--
GGTTTTTGT (recombination signal sequence),
or a slight variant of it, and each J segment is
preceded by ACAAAAACC--spacer--CACTGTG,
or a slight variant of it.
This is true for the variable regions and
J segments associated with heavy chain, kappa,
and lambda light chain genes.

32. orange arrow: CACAGTG
blue arrow: GGTTTTTGT
Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997

33. V(D)J recombination is mediated by the
lymphoid specific recombination activating genes
RAG1 and RAG2.
Mutations in the recombination activating genes
lead to severe combined immunodeficiency (no B
or T cells) or to Omenn s syndrome, a milder
immunodeficiency.

34. Summary
1. Innate immunity involves recognition by TLRs and
NODs--receptors for pattern molecules.
2. NK cells kill cells with low expression of MHC class I
molecules.
3. Antigen-independent B cell differentiation occurs in
the bone marrow.
It includes several stages that involve changes in
immunoglobulin heavy and light chain expression, as
well as other CD antigens.
Antigen-independent B cell differentiation results in a
repertoire of at least ten million clones of B cells.

35. 4. Anti-self, immature B cells are deleted.
5. Variable and constant region genes for
immunoglobulins are separated in DNA.
6. Variable regions are encoded by many genes
—Germline diversity.
7. V(D)J joining uses specific sequences
(CACAGTG--spacer--GGTTTTTGT) and is
mediated by the recombination activating gene
products.

36. Additional Source Information
for more information see: http://open.umich.edu/wiki/CitationPolicy
Slide 12: University of Michigan Department of Microbiology and Immunology
Slide 17: Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997
Slide 18: Minegishi et al., J. Clin Invest. 104: 1115-21 (1999)
Slide 20: Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997
Slide 23: Regents of the University of Michigan
Slide 25: Regents of the University of Michigan
Slide 28: Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997
Slide 32: Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997