Teo TP T cell devo BSSº ano... · X-linked Severe Combined Immunodeficiency Human autosomal recessive SCID can have many genetic causes, like mutations in the genes encoding the

04-10-2011

1

The differentiation of B cells

BONE MARROW SECONDARY LYMPHOID ORGANS

? pro BpreB I

preB II

immature B mature B plasma cellPrecursor

antigensecondary signals

class switchantigenprimary signals

activated B cell

(T cell help)

Funções dos anticorpos:

Os linfócitos B produzem anticorpos;

Os anticorpos reconhecem antigénios “inteiros”

Figure 3-11

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GATA-1-

GATA-2++

Aiolos-

GATA-1-

GATA-2-

Aiolos+

GATA-3+

GATA-1+

GATA-2+

Aiolos-

GATA-3-

PU.1- GATA-3++

PU.1++ GATA-3-

IL-7R-

Epo-R+

IL-7R+

Epo-R-

HSC

CLP

CMP

GMP

MEP

pro-T

pro-B

T cell

NK cell

B cell

Granulocyte

Megakaryocyte

Erythrocyte

Monocyte

T celldevelopment

CommonLymphoidProgenitor

- Objectivo?

Produzir células dotadas de um TCR funcional, e por isso capazes de

reconhecer antigénio e montar uma resposta imunitária celular.

- Onde e quando?

No timo do adulto (e no fígado do feto).

(Notar diferença em relação a células B: medula óssea)

- Como?

Através da diferenciação a partir de células pluripotentes,

as quais sofrem alterações específicas de expressão génica,

as quais determinam a linhagem T.

timo

“Desenvolvimento-T”

T cell receptor (TCR):

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TCR Rearrangements

TCR β locus - Germline

(52)V Regions J1(1-6)D1 C1 D2 J2(1-6) C2

1. D to J Rearrangement

2. V to DJ Rearrangement

4. mRNA SplicingTCRβ Protein

3. TranscriptionmRNA

T Cells are defined by the T Cell Receptor

ss

ss

ss

ss

s-s

ITAM (Immunoreceptor tyrosine-based activation motif)

CD3δCD3εCD3ε CD3γ

CD3ζ CD3ζ

ss

ss

ss

ss

s-s


CD3ζ CD3ζ

ss

ss

ss

ss

TCRβ TCRα

αβ T Cell Receptor

ss

ss

ss

ss

TCRγ TCRδ

γδ T Cell Receptor

Survivalcycling

SurvivalActivationDeath

Development

(antigen-independent)

in Bone Marrow

Activation & Differentiation

(antigen-dependent)

in peripheral lymphoid organs

Pre-B cell B lymphocyte

5,

3,

VpreB λλλλ5

(Human chromosome 22, mouse 16)

Light chainVpreB

λλλλ5

VpreB

λλλλ5

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Figure 7-2

A estrutura do timo adulto

Cortex

Medulla

Cortical Epithelial Cells

Medullary Epithelial Cells

Macrophages

Dendritic Cells

Figure 7-9

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Figure 7-10 part 1 of 3

WT mice Nude mice

Mutação do gene foxn1 , factor deTranscrição fundamental para adiferenciação das células epiteliais.

No thymus, no T cells!

- Thymectomy at birth

- DiGeorge’s syndrome (humans) - Chr 22

Clinicians

José Gonçalo MarquesSusana Lopes da SilvaMaria João RodrigoCatarina Nascimento Ana Carvalho

Imunologia Clínica, IMM / FML

Ana Espada de SousaAdriana AlbuquerqueRui M. M. Victorino

Duke University Medical Center, Durham, NC, USALouise Markert

Pediatric Immunology Unit, Frederico II University, Naples, Italy

Claudio Pignata

Unité des Virus Lents, Institute Pasteur, Paris, Fr anceRemy Cheynier

Imunologia Clínica IMM/FML

Nude/SCID: a clinical case at HSMNude/SCID: a clinical case at HSM

� 3 months old

� BCG adenitis + mild dermatitis

� Alopecia totalis + nails distrophy

Clinical HistoryClinical History


� 5 months old

• admitted because of severe respiratory

failure that required mechanical ventilation

Athymia

� Micobacterium bovis dissemination

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Severe combined Immunodeficiency

“SCID”

T lymphopenia+

Absence of circulating naive T cells+

oligoclonal expansion of activated memory T cells+

Major impairment of lymphoproliferative responses to mitogens and antigens including PPD+

Hipogammaglobulinemia

Immunological presentation + total alopecia + nails distrophy → nude SCID/FOXN1 deficiency


A homozygous C-T transition at nucleotide position 792 of FOXN1 gene was detected in the patient

Mother

T C

Father

T C

Patient

Genetic DiagnosisGenetic Diagnosis


Thymus transplantationThymus transplantation

Thymus of the donor

Culture in the presence of deoxyguanosine during

12-21 days to deplete the hematopoietic cells

The thymus slicesare transplantedinto the infant‘squadriceps.

Biopsy performed 2 months after transplantation confirmed viability

of the thymic tissue and the presence of Hassall’s bodies.

HLA-A HLA-B HLA-C HLA-DRB1 HLA-DQB1

2601 3503 1203 0701 0202 Patient

3101 3801 1201 0301 0101 0801 0701 1101 0301

Donor 2301 4901 1301 0603

The only HLA sharing was in HLA-DQB1*0301.

14 months old


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Immunological reconstitutionImmunological reconstitution

Thymic transplantation was associated with a slow a nd progressive recovery of CD4 + T-cells, maintaining low T-cell counts.

Months post-transplantation

0 5 10 15 20 25 30

0

5

10

15

20

25

30

35

% o

f lym

phoc

ytes chickenpox

prednisolone

ciclosporine

0 5 10 15 20 25 30

0

100

200

300

400

500

600

700

CD4+CD8+

cells

/µl

chickenpox

prednisolone

ciclosporine

Strikingly, the recovery of the CD8 + T-cell pool was not as marked.


ConclusionConclusion

� This is the first report of thymic transplantation in a patient with FOXN1 deficiency.

� A slow progressive immune reconstitution after thymic transplantation was observed although the CD8 recovery was much less marked than the CD4 T-cell subset.

� The child remains at home and free from infections for more than 2 years.


T cell development

THYMUS

Pro-T Pre-T

TCRγδγδγδγδ +

CD4- CD8-(DN)

bonemarrowprecursor

Marcadores:Proteinas de superfície,

detectadas por anticorpos

CD4+ SP (helper)

CD8+ SP (cytotoxic)

CD4+CD8+(DP)

TCRαβ αβ αβ αβ +

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Como identificar populações de timócitos?

CD8

CD4

DN

DP

SP8

SP4

Marcadores

Anticorposfluorescentes

FACS(Fluorescence-AssociatedCell Sorting)

16%

TCRγδγδγδγδ

γδ γδ γδ γδ

CD44

CD25

DN

1 2

34

DN1 DN2 DN3 DN4 DP

γδγδγδγδ thymocytes

Receptores para sinais de diferenciação-T

IL-7R

The Role of IL-7 in T Cell Development

1 3CLP

Thymus

2 DP

γδ T Cell

4

DN (especially DN2) thymocytes express IL-7R

Mice without IL-7(R):

10-fold reduced numbers

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X-linked Severe Combined Immunodeficiency

Human autosomal recessive SCID can have many genetic causes,like mutations in the genes encoding the ADA and PNP enzymes,or ZAP-70 and Jak-3 kinases (signal transducers for TCR/ cytokines).

The X-linked SCID (only affects males) is caused by mutations inthe gamma chain of the IL-7 receptor (IL-7R), also known as thecommon gamma chain (γc, shared with IL-2R/4R/9R/15R/21R).

SCID is usually only detected after serious opportunistic infection.

Clinical indication: Absence of T cells, normal B cell count.

Specific test: Staining B cells with anti-γc monoclonal antibody.

Therapy: Bone marrow transplantation (from healthy, compatible donor).

DN1 DN2 DN3 DN4 DP


preTCR


IL-7R

The Pre-T Cell Receptor & “β-Selection”

ss

ss

ss

ss

s-s

ss

ss

ss

ss


TCRβ TCRα


TCRζ TCRζ

ss

pTαααα


ss

ss

ss

ss

s-s

ss

ss

TCRβ

CD3δ/γCD3εCD3ε CD3γ

TCRζ TCRζ

Pre-T Cell Receptor

pTαααα (preTCRα):α):α):α):

Produto de gene que

não rearranja (≠TCR)

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Consequences of preTCR selection:

DN

1 3CLP

Thymus

2

WithoutpreTCR

DN

1 3CLP

Thymus

2

SURVIVAL

4 DP

DIFFERENTIATION

PROLIFERATION

Consequences of preTCR selection:

VDJ IgH

VDJ γγγγ, , , , ββββVJ δδδδ

Rag

Rag

VJ IgL

VJ αααα

Rag

Rag

ααααββββ T

γγγγδδδδ ΤΤΤΤ

pro-lymphoid

pre-pro-B pro-B pre-B I

(preBCR) pre B II

preTCR

B

BCR

α/β α/β α/β α/β TCRT

γ/δγ/δγ/δγ/δTCR

T

ParaleloT – B:

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Figure 7-22Outras proteínasimportantes nodesenvolvimento-T:

DN1 DN2 DN3 DN4 DP


preTCR TCR


Notch,IL-7R

TCR versus preTCR

ss

ss

ss

ss

s-s

ss

ss

ss

ss


TCRβ TCRαααα


TCRζ TCRζ

ss

pTαααα


ss

ss

ss

ss

s-s

ss

ss

TCRβ

CD3δ/γCD3εCD3ε CD3γ

TCRζ TCRζ

Pre-T Cell Receptor

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Major Checkpoints in αβ T Cell Development

Helper T Cell

Cytotoxic T Cell

DN

DP

CD4 SP

CD8 SP

1 2 3 4CLP

Thymus

TCRβTCRγ RearrangementsTCRδ

Checkpoint 1preTCR selection

TCRααααRearrangements

Checkpoint 2TCR selection

MHC

TCRββββTCRαααα

TCRαβ binds MHC-peptide complexes

- two large sets of very diverse molecules!

MHCClass I

T cell

APC

Peptide

Positive & Negative Selection:TCR binds to MHC+peptide

CD4 or CD8Co-Receptor

Thymic AntigenPresenting Cell

MHC Class I or Class II

βα

DP ThymocyteTCR

Self-peptide

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Recognition of MHC class II

Positive selection: from DP to SP (CD4+ or CD8+) th ymocytes

Recognition of MHC class I

downmodulation of CD4 expression

CD8+ T cells

downmodulation of CD8 expression

CD4+ T cells

• no MHC class I:absence of CD8+ T cells

• no MHC class II:absence of CD4+ T cells

Positive selectionoccurs on cortical

epithelial cells

Negative selection of thymocytes on MHC + self pept ide

weak interaction intermediate interaction

strong interaction

no stimulusdeath

stimulusto proliferate

death by apoptosis

X X

• Negative selection is required to eliminate T cells that interactstrongly with MHC + self peptide

• Negative selection mainly occurs on bone marrow der ived DCs and macrophages present in the thymic medulla

• most thymocytes (circa 95%) die during development i n the thymus

CD4+ orCD8+ T cell

Avidity of TCR/MHC Interaction controls Positive and Negative Selection

Avidity of T Cell ReceptorMHC interaction

Low Avidity Medium Avidity High Avidity

Death(Neglect)

Positive Selection Negative Selection(Death)

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AIRE promotes central tolerance

Tolerance: - Central: depletion in the thymus (Neg.Sel)

- Peripheral: control by “regulatory T cells”

Problem: how to deplete, in the thymus, T cells recognising

tissue-specific antigens?

Mutations in AIRE cause autoimmune polyendocrinopathy syndrome type 1 (APS-1): chronic mucocutaneous candidiasis, multiple autoimmune endocrinopathies, ectodermal dystrophies.

AIRE (Autoimmune regulator) controls the expression of tissue-specific proteins in the thymus!

Os diferentes estadios de maturação medular dos linfócitos B

reflectem diferentes fases do processo de recombinação somática V(D)J

Desenvolvimento-B

V(D)J Não Iniciados p H Completados p H Completados p L

HSC/ CLP pró-B pré-B B

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In mature B cells (after VDJ rec) additional genome modifications:Further diversification of variable region and Isot ype switching

Variable region Effector (conserved) region

região constante

região variávelx2

ligação ao Ag

função efectora

5 tipos de região C

das cadeias pesadas

5 isotipos de

Anticorpos,

com funções distintas

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The first antibodies produced are of the IgM isotype.

To get different Ab functions, isotype must switch!

Changes the effector part, but NOT the variable region of the antibody!

IgM IgD IgG3 IgG1 IgG2b Ig2a IgE IgA

Class switch, in mature B cell upon activation A recombination event of the effector region:

e.g. IgM to IgG1

Documents

Teo TP T cell devo BSSº ano... · X-linked Severe Combined Immunodeficiency Human autosomal recessive SCID can have many genetic causes, like mutations in the genes encoding the