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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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


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


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.


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)




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

Maturation


Today distinct parts of the thymus

Thursday



Most developing thymocytes die in the thymus distinct parts of the thymus

apoptotic cells

(DNA fragmentation)

macrophages

Macrophages...garbage collectors, contain the dying cells


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


TCR rearrangement is very similar to Ig rearrangement assembly and testing of the newly rearranged TCR


Figure 5 6 part 1 of 3

First step: TCRbeta rearrangement assembly and testing of the newly rearranged TCR

Figure 5-6 part 1 of 3


Figure 5 6 part 2 of 3
Figure 5-6 part 2 of 3 assembly and testing of the newly rearranged TCR


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 if you have not rearranged TCRalpha? 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


Does the pre-TCR have a ligand? if you have not rearranged TCRalpha?

Remove extracellular domains…

…and examine T cell development

(work of Nigel Killeen, UCSF)


Pre-TCR extracellular domains are not required! if you have not rearranged TCRalpha?

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

(Irving (1998) Science 280:905)


CD25 if you have not rearranged TCRalpha?

b-selection leads to proliferation

DN2

Gated on CD4/CD8 DNs

DN3

Proliferating cells are bigger

Forward scatter

(cell size)


TCR if you have not rearranged TCRalpha?b locus has two D-J clusters

Allows a 2nd rearrangement if 1st is nonproductive


We are here if you have not rearranged TCRalpha?


Progression through development if you have not rearranged TCRalpha?

correlates with rearrangement


At the DP stage, TCRalpha rearrangement begins if you have not rearranged TCRalpha?


TCR if you have not rearranged TCRalpha?a locus has 50 Ja segments


TCR if you have not rearranged TCRalpha?a locus-- replacement rearrangement


Dp thymocytes express surface tcr
DP thymocytes express surface TCR if you have not rearranged TCRalpha?

CD3 lo cell-- pre-TCR

CD3 hi cells-- TCR


Tcr chain is required for dp to sp transition experimental evidence
TCR if you have not rearranged TCRalpha? 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


Differential expression of Src family kinases if you have not rearranged TCRalpha?


Remember what lck does
Remember what Lck does if you have not rearranged TCRalpha?


Role of Lck in T cell development if you have not rearranged TCRalpha?

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!


 if you have not rearranged TCRalpha? vs 

Thursday


A model for if you have not rearranged TCRalpha? versus  lineage commitment


TCR if you have not rearranged TCRalpha? rearrangement removes the delta locus


TCR if you have not rearranged TCRalpha? lineage comprises the majority of T cells

90%

10%


gd if you have not rearranged TCRalpha?T cells are favored

during early fetal development


Waves of if you have not rearranged TCRalpha? T cells with specific TCR usage

develop early gestation


Waves of if you have not rearranged TCRalpha? T cells home to different tissues


gd if you have not rearranged TCRalpha? 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


Antigen recognition by if you have not rearranged TCRalpha? 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


Dendritic epidermal T cells (DETC) if you have not rearranged TCRalpha?

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

Involved in wound healing as well as tumor protection


Detcs promote wound healing
DETCs promote wound healing if you have not rearranged TCRalpha?


Roles of if you have not rearranged TCRalpha?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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