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B Cell Development

M1 – Immunology Sequence

Winter 2009

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?

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.

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

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.

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.

NOD receptors are another set of receptors associated with innate immunity. They are expressed in the cytoplasm.

NOD: containing a nucleotide-binding/ oligomerization domain

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.

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).

Stem cell Pro B cell Pre B cell Immature B cell

Mature B cell

Absent Absent µ chain in cytoplasm

IgM on cell surface

IgD and IgM on cell surface

µ

Y Y Y

University of Michigan Department of Microbiology and Immunology

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).

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.

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.

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

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.

Normal Immunodeficient Immunodeficient

Pro and Pre B cells B cells

Stem cells

Minegishi et al., J. Clin Invest. 104: 1115-21 (1999)

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).

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

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.

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).

Each antibody is made by one clone of B cells. Hence, antibodies are “clonally distributed”.

Regents of the University of Michigan

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

Regents of the University of Michigan

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.

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.

Vκ encodes amino acids 1-95 Jκ encodes amino acids 96-107 Cκ encodes amino acids 108-214

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

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.

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.

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. .

orange arrow: CACAGTG blue arrow: GGTTTTTGT

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

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.

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.

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.

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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