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T Cell Development I: The Generation of TCR+ Thymocytes

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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

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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

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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.

Nude Mice

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scid mutation: DNA PKno gene rearrangement

nude mutation: transcription factor required for terminal epithelial differentiation(whn)

NO B or T CELLS NO THYMUS

The Thymus is required for T cell development

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T cell development occurs in the thymus

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Figure 5-3 part 1 of 2

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The epithelial cells & developing thymocytes

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T cell development can be characterized using flow

cytometry

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Real data

CD

4

CD8

8 88

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Double-positives

Double-negatives

CD4 single-positive

CD8 single-positive

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DN’s can be further subdivided into DN1 through DN4

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

CD25 (chain of IL2R)

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The real data

CD25

(gated on DN cells)

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Double negative cells appear in the fetal thymus before double positive cells

K. Hogquist

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T cell populations during development and in the lymphoid tissue

8.383.24.711.47.9

CD4

CD8

THYMUS SPLEEN

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

Maturation

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Today

Thursday

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Most cells fail to complete thymocyte maturation

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macrophagesapoptotic cells

(DNA fragmentation)

Macrophages...garbage collectors, contain the dying cells

Most developing thymocytes die in the thymus

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

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TCR rearrangement is very similar to Ig rearrangement

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Figure 5-6 part 1 of 3

First step: TCRbeta rearrangement

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Figure 5-6 part 2 of 3

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Pre-T cell receptor

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

rearranged TCRalpha?Pre-Talpha

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TCR chain is required for DN to DP transition:Experimental Evidence

WT TCR-/- TCR-/- TCR/-/- TCR/-/-

ExpressingTCR

108 108 106<107 <107

CD4

CD8

Totalthymocytenumbers

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Remove extracellular domains…

…and examine T cell development

(work of Nigel Killeen, UCSF)

Does the pre-TCR have a ligand?

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pTT;T receptor restores CD4+8+ development and thymocyte expansion

(Irving (1998) Science 280:905)

Pre-TCR extracellular domains are not required!

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CD25

Forward scatter(cell size)

Proliferating cells are bigger

-selection leads to proliferation

DN2

DN3

Gated on CD4/CD8 DNs

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TCR locus has two D-J clusters

Allows a 2nd rearrangement if 1st is nonproductive

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We are here

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Progression through development correlates with rearrangement

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At the DP stage, TCRalpha rearrangement begins

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TCR locus has 50 J segments

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TCR locus-- replacement rearrangement

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DP thymocytes express surface TCR

CD3 lo cell-- pre-TCRCD3 hi cells-- TCR

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TCR chain is required for DP to SP transition:Experimental Evidence

WT TCR-/- TCR-/- TCR/-/- TCR/-/-

ExpressingTCR

108 108 106<107 <107

CD4

CD8

Totalthymocytenumbers

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Differential expression of Src family kinases

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Remember what Lck does

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0.4 x 106 288 x 106 10 x 106Totalthymocytenumber

Role of Lck in T cell development

RAG KORAG KO

TCRTransgeneRAG KO x Lck KOTCRTransgene

CD

4

CD8

30-fold reduction in DPs!

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Thursday

vs

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A model for versus lineage commitment

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TCR rearrangement removes the delta locus

4310%90%

TCR lineage comprises the majority of T cells

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T cells are favored during early fetal development

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Waves of T cells with specific TCR usage develop early gestation

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Waves of T cells home to different tissues

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• cells bearing specific receptors end up in skin (V5), gut (V2),

uterus (V6), etc.

• Limited junctional diversity: no N nucleotides (no TdT)

• The mechanisms controlling this limited rearrangement are poorly

understood.

Early waves of T cells

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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

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Dendritic epidermal T cells (DETC)

V5 T cells home to the skin and wedge among keratinocytes

Involved in wound healing as well as tumor protection

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DETCs promote wound healing

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Roles of T cells in cancer

• -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

Girardi et al, Science 294:605 (2001)

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