the development of b lymphocytes - universiteit...

The Development of B lymphocytes Chapter 6 Parham

Hans de Haard 21th of May 2010

Agenda

•  Stages in development of B cells

•  B cell development in bone marrow

•  Heavy chain rearrangement

•  Light chain rearrangement more efficient process

•  Checkpoints

•  B1 versus B2 lymphocytes

•  Removal self reactivity

•  B cell development in secondary lymphoid tissues

•  Differentiation into mature B lymphocytes

•  Maturation into plasma cells and memory cells

•  Various B cell tumors represent different stages in B cell development

Stages in development B cells

•  Phase 1: B cell precursors in bone marrow acquire

functional antigen receptors via gene rearrangements

•  Phase 2: elimination anti-self (tolerance) in bone

marrow and 2nd lymphoid tissues

•  Phase 3: positive selection for B cells expression of

pathogen specific Ig in 2nd lymphoid tissues

•  Phase 4: mature B cells circulate via lymph and blood

to lymphoid tissues patrolling for infections

•  Phase 5: proliferation and clonal expansion pathogen

specific B-cells in 2nd lymphoid tissues

•  Phase 6: maturation into:

•  plasma cells =>remain in 2nd lymphoid tissues, express large quantities

pathogen specific Ab

•  memory B cells (circulate) for rapid Ab generation in case of future

infections with same pathogen

Agenda





•  Checkpoints







Development Immature B cells in bone marrow

•  B and T cells originate from common lymphoid progenitor cell (CD34+

pluripotent hematopoietic stem cell)

•  Earliest B cell lineage is early pro-B cell with starting DH – JH rearrangements

•  Late pro-B cell VH rearranges with DJ, in pre-B cell stage VL with JL

•  In immature B cell stage IgM expressed on surface

Heavy chain rearrangement inefficient process •  Due to imprecise joining and addition of N / P nucleotides during VDJ

recombination (3 vs 2 segments for light chain) high probability for incorrect

reading frame yielding non-functional Ab => non-productive rearrangement

•  Check for correctly expressed heavy chain; if not, then rearrangement of

other chromosome

•  If again non-productive arrangement, then cell dies by apoptosis

•  Just over half of total number of B cells make functional heavy chain

Quality check (pre-) B cell receptor

•  Correctly rearranged m heavy chain capable to

combine with light chain

•  Check performed in ER with surrogate light chain

consisting of VpreB and l5 in pre-B cell receptor

•  IgM incorporated in cell membrane via MC, but

associated Iga / Igb responsible for signaling

•  Final check with rearranged light chain in

B cell receptor expressed on cell membrane

•  After successful VDJ recombination on first chromosome assembled pre-B

cell receptor signals to stop recombination of 2nd chromosome (allelic

exclusion)

Light chain rearrangement more efficient process

•  Large pre-B cell with rearranged heavy

chain divides yielding approx. 100 cells

per B cell clone (i.e. with identical VDJ)

•  Kappa light chain rearranges first, if

unsuccessful followed by lambda

•  Only V and J need to recombine, therefore

higher success rate then with additional D

•  If first recombination fails to give correct

fusion, then other V region in upstream position will recombine with downstream J

•  If again non-productive, then recombination of kappa locus on other chromosome

•  If kappa fails, then lambda locus rearranges

•  85% of pre-B cells yield productive light chain rearrangement

•  Check (1): pre-B cell receptor with correct VDJ fused to m heavy chain and

associated with surrogate light chain (in vesicle)

•  Check (2): B cell receptor with correctly rearranged light chain and m

heavy chain (on cell membrane)

Checkpoints during B cell development in bone marrow

Regulated and time dependent expression of proteins involved in Ig rearrangement

•  Expression of signaling proteins

important for differentiation and

development of B cell:

•  FLT3 => receptor kinase, receives

signal stromal cells for differentiation

of common lymphoid progenitors

•  CD19 => co-receptor participating in

Ag – BCR complex

•  CD45 => receptor phosphatase

modulating signals from Ag – BCR

complex

•  RAGs are expressed two times,

during rearrangement of heavy

and later during light chain locus

Pax-5 opens up chromatin in heavy chain locus: start recombination

•  Pax-5 is B cell specific transcription

factor

•  Binds enhancer elements downstream

(i.e. 3’ direction) of constant region

•  Thereby opens up chromatin resulting

in low level transcription from

promoters upstream (5’) of D and J

•  Transcription allows access of RAGs to

“open” HC locus to start

recombination of D – J

•  Brings enhancer closer to V region

leading to transcription from

promoters upstream of V

•  Induces V recombination with DJ

B1 versus B2 lymphocytes

•  Minority human B cells are of B1 class

=> CD5 marker typical for T cells

=> little or no surface IgD

•  Arise from stem cell active in

prenatal period and therefore less

developed

=> use V genes closely positioned to D

=> no or limited TdT activity (i.e. few

N nucleotides introduced)

•  Low affinity antibodies and polyspecific (i.e. reactive against multiple

antigens), often carbohydrates recognized

•  When generated in postnatal period these are better developed and matured;

no longer produced by bone marrow, but are self-renewal at sites in

peripheral circulation (IL-10 dependent)

•  Chronic lymphocytic leukemia caused by B1 cells

Summary B cell development in bone marrow

Removal self-reactive antibodies

•  Self-reactive immature B cells

removed

=> 75% total population

•  Potential multivalent self antigens

are glycoproteins, proteoglycans

and glycolipids on cells

•  Negative selection starts in bone

marrow on self antigens

presented by stromal and

hematopoietic cells

•  Immature B cells with self

reactivity are maintained within

bone marrow, others will go into

circulation

Receptor editing for changing self reactivity

•  Self reactivity detected in bone

marrow

=> binding to mutivalent

antigen

=> signaling to reduce

surface expression IgM

•  Maintaining RAG expression

=> excision old light chain

=> replaced by newly recombined

light chain

(receptor editing)

•  If self reactivity has been removed,

then B cell development continues

•  If not, B cell in apoptosis

(clonal deletion)

Self reactivity against monovalent antigen results in anergy

•  Self reactivity detected in bone marrow

=> binding to monovalent antigen

=> B cells inactivated and unresponsive

against antigen (Anergy)

•  Anergic cells:

=> no production cell surface exposed IgM

(remains intracellular)

=> only IgD presented (not activating)

=> go into circulation, short T½ (1 – 5 days vs 40

days for other B cells)

•  Central tolerance: check within bone marrow

leading to removal of anti-self or anergy

•  Peripheral tolerance: removal of anti-self outside

bone marrow (i.e. by apoptosis or anergy)

Agenda





•  Checkpoints







Maturation B cells in secondary lymphoid tissues

•  Immature B cells developed in bone

marrow recirculate between blood and

secondary lymphoid tissues

•  Development from immature (sIgM, IgD)

into mature B cells (sIgM, IgD) occurs

within secondary lymphoid tissues:

=> enter lymph nodes through walls

High Endothelial Venules (HEV) driven

by chemotaxis (gradient of chemokines)

=> guided by other chemokines to primary lymphoid follicles, i.e. network of

Follicular Dendritic Cells (FDC)

=> contact FDC and B cells; if no encounter with antigen B cell detaches and

goes back in circulation via lymph

•  Now developed into mature naive B cell; survival dependent on regular passage

through primary follicles of secondary lymphoid tissues; long T½ (~100 days)

•  Encounter of mature naive B cell

with antigen leads to interaction

with antigen specific TH:

activation leads to proliferation and

differentiation into plasma cells

•  Change of phenotype: => expression of secreted IgM ( sIgM-) => not sensitive for signaling, not dividing

Differentiation of activated B cells into plasma cells

(picture lymph node see p 210)

• Migration to primary follicle thereby

becoming secondary lymphoid follicle with germinal centers

•  B cells become large proliferating centroblasts, subsequent

maturation into more slowly dividing centrocytes (isotype

switch and affinity matured)

•  As primary response subsides differentiation in memory cell

Various B cell tumors reflect different development stages of B cells

•  Mistake in rearrangement can lead

to activation of oncogenes

(controling cell growth, division and

differentiation)

•  B cell tumors are clonal, i.e. derived

from single recombined B cell

•  B cell tumors represent different

stages of B cell development

•  Tumors derived from naive B cells

grow in follicles of lymph nodes

=> follicular center cell lymphoma

•  Tumors derived from plasma cell

tumors grow in bone marrow

=> myeloma

Summary B cell development

The Development of B lymphocytes Chapter 6 Parham

Hans de Haard 21th of May 2010

the development of b lymphocytes - universiteit...

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