7 - lymphocyte development

errors in lymphocyte development result in:

• immunodeficiencies

• infectious diseases

• autoimmune disease

• lymphoma

general steps of lymphocyte development

precursor SC → mature properties gained → precursor properties lost

where do lymphocytes develop?

lymphoid tissues

B cell development in primary/central tissues

1. start in fetal liver and neonatal spleen

2. move to bursa + BM

T cell development in primary/central tissues

1. start in cryptopatches in intestine

2. move to thymus

lymphocyte maturation in peripheral/secondary tissues

spleen

lymph nodes

mucosal + cutaneous lymphoid tissues

is rearragement of Ag receptors reversible?

no

full lymphocyte development

1. HSC

2. MPP

   1. CFU - GEMM

   2. CLP - NK, B, T

B cell development

1. B cell precursor undergoes H + L recombination in BM

2. immature B cell negative selection in BM

3. mature B cell activates when bound to Ag in lymph organs

4. activated B cells differentiate in BM

negative selection

immature B cell binds to self Ags = apoptosis

types of active B cells

plasma and memory

stromal cells

in BM, provide signals that direct development of B cells

stromal cell examples

Flt3 ligand, CXCL12, IL-7, SCF

steps in B cell development

1. MPP

2. CLP

3. early pro-B

4. late pro-B

5. pre-B

6. immature B

B cell development in detail

1. MPP = Flt3 → Flt3 ligand

2. CLP

3. early pro-B = Kit → SCF

4. late pro-B

5. pre-B = recombines H + L → forms IgM

6. immature B

IL7

common lymphoid to pre-B

• delivers signals to promote survival + proliferation

CXCL12

recognized by CXCR4

• CXCL12→CXCR4 retains developing B in BM

antigen independent stages of B cell develpment

MPP → pre-B

expression of receptors is controlled by ______ in developing B cells

transcription factors

transcription factors of B cell development

Ikaros, PU.1, E2A, EBF, Pax5

PU.1 and Ikaros

induce expression of receptor Flt3 in MPP

induced expression of IL7 receptor induces _____

E2A → works w/ PU.1 and IL7

EBF

induced by PU.1 and IL7

• commits to B cell lineage

Pax5

induces expression of proteins that mark pro-B cell stage!

proteins that mark pro-B cell stage

CD19, BLNK and Ig alpha

• means H-chain recombination will start

rearragement of Ig genes in B cell development

stem cell

H + L chains = germline

surface Ig = none

rearragement of Ig genes in B cell development

early pro-B

H chain = D-J rearrage

L chain = germline

surface Ig = none

rearragement of Ig genes in B cell development

late pro-B

H chain = V-DJ rearrage

L chain = germline

surface Ig = none

rearragement of Ig genes in B cell development

large pre-B

H chain = VDJ rearraged

L chain = V-J rearranging

surface Ig = mu chain on surface

rearragement of Ig genes in B cell development

small pre-B

H chain = VDJ rearraged

L chain = V-J rearranging

surface Ig = intracellular mu chain

rearragement of Ig genes in B cell development

immature B cell

H chain = VDJ rearraged

L chain = VJ rearranged

surface Ig = IgM on surface

rearragement of Ig genes in B cell development

mature B cell

H chain = VDJ rearraged

L chain = VJ rearranged

surface Ig = IgM and IgD on surface

how is IgD expressed?

alternative mRNA splicing! not recombination

RAG1 + 2

VDJ recombinationT

Tdt

n-nucleotide addition to recombined H-chain

lambda 5 and VpreB

encode surrogate L-chain while its not in recombination

Ig alpha + beta, CD45R, Btk and CD19

signal transdction

CD45R and CD19

expressed on surface from late pro-B → mature

• distinguish B-lineage from other cell types in blood + lymphoid tissues

• staining reagents specific for CD19 or CD45R

what rearragement is essential for pre-B cell development?

V_H-DJ_H rearragement

3n coding sequence

Corresponding protein will be translated + synthesized as Mu chain of H chain

3n+1 or 3n+2 coding sequence

no reading frame, so no protein

what pairing indicates a pre-B cell

H-chain pairing with L-chain w/ VpreB and lambda 5

the pre-B cell receptor is composed of

mu chain and surrogate light chain

what does pre-BCR signaling require

Ig alpha and Ig beta proteins

BLNK and Btk

Bruton’s X-linked agammaglobulinemia

Btk mutation or deletion

pre-BCR signal transduction

1. clonal expansion

2. allelic exclusion = stops H-chain recombination

3. L-chain rearragement

what rearragement is essential for immature B cell development?

L-chain

productive L-chain recombination for B cells

L-chain synthesized

• can pair w/ H-chain

• will form surface IgM

non-productive L-chain recombination for B cells

NO L-chain, pre-B will undergo apoptosis

how many times can the kappa light chain be recombined?

5 because there are 5 kappa gene segments

how many productively rearranged Igs does a B cell express?

1

allelic exclusion of pro-B cell

H-chain recombination must be productive to proceed to pre-B

• tries on both alleles

  • success = pre-BCR on surface

  • unsuccessful = cell death

what does the expression of surface pre-BCR do?

transduces signals to STOP H-chain gene recombination

• down regulates RAG1 and RAG2

pre-B cell allelic exclusion

L-chain recombination must be productive to proceed to pre-B

what indicates an immature B cell

L chain pairs w/ H chain pairs w/ mu chain → surface IgM

immature B cells deliver signals to stop expressing ______

RAG1 and RAG 2

• inhibits light chain recombination

fate of immature B cells depends on _____________

binding avidity w/ self Ags

immature B cell w/ no self Ag reaction

migrates to periphery → mature B cell

multivalent central immature B cell

very high self affinity → receptor editing or apoptosis

soluble central immature B cell

moderate self affinity →

migrates to periphery →

down regulation of IgM →

anergic B cell (no response to Ag)

low-affinity central immature B cell

can no longer respond to Ags →

migrates to periphery →

matures

why are low-affinity B cells considered “seeds of autoimmunity”

they may be activated + respond to their self Ags

replacement of Ig light chains by ___________ can rescue some autoreactive B cells by changing their Ag specificity

receptor editing

why doesn’t receptor editing happen at the H-chain?

there are no available D segments

VH replacement

rare H-chain revision mechanism

• RAG can use signal sequences w/in rearraged VH to swap upstream VH segment, altering H-chain CDR3

central tolerance induction

• site: in central/primary tissues

• stage: immature lymphocytes

• primary mechanisms: negative selection, receptor editing

• role: prevents self Ag reactivity

peripheral tolerance induction

• site: in peripheral/secondary tissues

• stage: immature → mature

• primary mechanisms: negative selection, receptor editing

• role: prevents self Ag reactivity

multivalent peripheral immature B cell

apoptosis

soluble peripheral immature B cell

anergic → apoptosis

low-affinity peripheral immature B cell

mature B cell clonally ignorant

no self Ag peripheral immature B cell

mature B cell

T1 of B cells in spleen

express IgM + BAFF receptor

BAFF

principal B cell survival factor

T2 of B cells in spleen

express CD21

if T1 and T2 B cells DO NOT recognize self Ags where do they go

they mature into follicular B cells

marginal zone B cells

~10% upregulate CD21

• low IgD

• develop and STAY in marginal zone

follicular B cells

• very high IgD

• in secondary lymphoid tissues in B cell follicles

B1 B cells

arise early in development

• only abt 5%

• limited diversity

• in body cavities

• no T cell help

• do not go through germinal center

B2 B cells

over 90%

• marinal + follicular

• vast diversity

• need T cell help

• best at germinal center reax.

T cell development steps

1. developed progenitors migrate from BM → thymus

2. immature T cell goes through rigorous positive + negative selection in thymus

3. ones that survive selection mature

T cell development steps

1. progenitors migrate BM → thymus

thymic stromal cells = notch signaling to initiate recombination of T cell receptor genes

• commits progenitor to T cell development

• productive recombination → immature T cell + TCR on surface

T cell development steps

2. immature T cell positive + negative selection in thymus

TCR on surface can recognize self-peptide presented in complex

positive selection

T cell recieves survival signal

negative selection

interaction btw TCR + self-peptide MHC complex too strong

• apoptosis to prevent autoimmunity

T cell development steps

3. immature T cells that survive positive + negative selection can mature

can migrate through blood → peripheral tissues

T cell development in peripheral lymphoid tissues

if mature T cell encounters Ag by professional APC

• T cell recieves co-stimulatory signals provided by DC

• T cell proliferates + differentiates

T cell development in peripheral → infection

effector T cells clear infection

• CD8 = viral infection

• CD4 = activate macrophage to kill intracellular pathogen

thymic stroma

network of epithelial cells surrounding developing thymocytes

• provide Notch1 ligand for T cell lineage commitment

developing thymocytes that undergo apoptosis are ingested by __________ in the thymic cortex

macrophages

thymic stromal cells

thymic cortical epithelial cells + medullary epithelial cells

• form network epithelial cells surrounding developing T cells

alpha:beta T cells express ________

alpha:beta TCR

CD4 phenotype

CD3+CD4+CD8-

CD8 phenotype

CD3+CD4-CD8+

gamma:delta T cells express ________

gamma:delta TCR

gamma:delta phenotype

CD3+CD4-CD8

NKT cells phenotype

CD3+CD4-CD8-NK1.1+

CD3 is commonly used as a pan marker for __________

T cells

stages of T cell development correlate with _________ and the expression of __________

TCR gene rearragement, transcription factors

3 developmental stages of T cell development

1. double negative = no CD4 or CD8

2. double positive = both CD4 and CD8

3. single positive = either

where does recombination start in T cell development

DN3