t cell development i the generation of tcr thymocytes
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T Cell Development I: The Generation of TCR + Thymocytes. Questions for the next 2 lectures: How do you generate a diverse T cell population with functional TCR rearrangements? How do you generate a T cell population that is self-MHC restricted?

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slide2

Questions for the next 2 lectures:

How do you generate a diverse T cell population with functional TCR rearrangements?

How do you generate a T cell population that is self-MHC restricted?

How do you ensure that those diverse T cell receptors are not-self reactive?

How do you coordinate lineage specification with MHC specificity and coreceptor expression?

•  vs.  T cell

• CD4 vs. CD8

slide3

The pathway of T cell development

T cells...precursors are born in the bone marrow and “educated” in the thymus.

  • The thymus “involutes” with age
  • DiGeorge Syndrome, Nude mice
slide4

Nude Mice

A. Human (mammal) skin after 60 days.

B. Cat (mammal) skin at 51 days.

C. Chicken (bird) at 32 days;

D. Chameleon (reptile) at 41 days.

E. Fence lizard (reptile)at 28 days.

F. Tree frog (amphibian) at 40 days.

slide5

The Thymus is required for T cell development

scid mutation:

DNA PK

no gene rearrangement

nude mutation:

transcription factor required for

terminal epithelial differentiation

(whn)

NO THYMUS

NO B or T CELLS

real data

Double-positives

CD4 single-positive

88

8

CD4

Double-negatives

CD8 single-positive

1

3

CD8

Real data
dn s can be further subdivided into dn1 through dn4
DN’s can be further subdivided into DN1 through DN4

CD25 (a chain of IL2R)

“Gate” on CD4-CD8-, analyze for CD44 and CD25

the real data

CD25

The real data

(gated on DN cells)

slide16

Thymocytes at different developmental stages are found in distinct parts of the thymus

Maturation

slide17

Today

Thursday

slide19

Most developing thymocytes die in the thymus

apoptotic cells

(DNA fragmentation)

macrophages

Macrophages...garbage collectors, contain the dying cells

slide20

T cell development involves the sequential generation, assembly and testing of the newly rearranged TCR

slide24

How do you test for successful TCRbeta chain rearrangements if you have not rearranged TCRalpha?

Pre-Talpha

Pre-T cell receptor

tcr chain is required for dn to dp transition experimental evidence
TCR  chain is required for DN to DP transition:Experimental Evidence

TCRa-/-

TCRb-/-

TCRa/b-/-

TCRb/d-/-

WT

108

108

<107

<107

106

Total

thymocyte

numbers

Expressing

TCRgd

CD4

CD8

slide26

Does the pre-TCR have a ligand?

Remove extracellular domains…

…and examine T cell development

(work of Nigel Killeen, UCSF)

slide27

Pre-TCR extracellular domains are not required!

pTaT;bTreceptor restores CD4+8+ development and thymocyte expansion

(Irving (1998) Science 280:905)

slide28

CD25

b-selection leads to proliferation

DN2

Gated on CD4/CD8 DNs

DN3

Proliferating cells are bigger

Forward scatter

(cell size)

slide29

TCR b locus has two D-J clusters

Allows a 2nd rearrangement if 1st is nonproductive

slide31

Progression through development

correlates with rearrangement

dp thymocytes express surface tcr
DP thymocytes express surface TCR

CD3 lo cell-- pre-TCR

CD3 hi cells-- TCR

tcr chain is required for dp to sp transition experimental evidence
TCR  chain is required for DP to SP transition:Experimental Evidence

TCRa-/-

TCRb-/-

TCRa/b-/-

TCRb/d-/-

WT

108

108

<107

<107

106

Total

thymocyte

numbers

Expressing

TCRgd

CD4

CD8

slide39

Role of Lck in T cell development

RAG KO

TCRTransgene

RAG KO x Lck KO

TCRTransgene

RAG KO

CD4

CD8

Total

thymocyte

number

0.4 x 106

288 x 106

10 x 106

30-fold reduction in DPs!

slide44

gdT cells are favored

during early fetal development

slide47
gd cells bearing specific receptors end up in skin (Vg5), gut (Vg2), uterus (Vg6), etc.

Limited junctional diversity: no N nucleotides (no TdT)

The mechanisms controlling this limited rearrangement are poorly understood.

Early waves of  T cells

slide48

Antigen recognition by  T cells is different than  T cells

 T cells are not MHC restricted!

Antigen is recognized directly, more like an antibody

In some cases ligands for the  TCR are self proteins upregulated under stress conditions

In humans, circulating  cells recognize a phospholipid antigen from Mycobacterium tuberculosis

slide49

Dendritic epidermal T cells (DETC)

Vg5 gd T cells home to the skin and wedge among keratinocytes

Involved in wound healing as well as tumor protection

slide51

Roles of gd T cells in cancer

• gd-knockout mice have a higher incidence of skin cancers induced by carcinogens, including DMBA/TPA (tumor initiator/promoter) (shown in figure) or methylcolanthrene (MCA).

• The protection is mediated by DETC resident in the mouse skin

Appearance of tumor cells in knockout versus wildtype mice treated with carcinogen

Girardi et al, Science 294:605 (2001)

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