1 T Cell Development I: The Generation of TCR + Thymocytes.
-
date post
22-Dec-2015 -
Category
Documents
-
view
222 -
download
1
Transcript of 1 T Cell Development I: The Generation of TCR + Thymocytes.
![Page 1: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/1.jpg)
1
T Cell Development I: The Generation of TCR+ Thymocytes
![Page 2: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/2.jpg)
2
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
![Page 3: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/3.jpg)
3
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
![Page 4: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/4.jpg)
4
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
![Page 5: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/5.jpg)
5
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
![Page 6: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/6.jpg)
6
T cell development occurs in the thymus
![Page 7: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/7.jpg)
7
Figure 5-3 part 1 of 2
![Page 8: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/8.jpg)
8
The epithelial cells & developing thymocytes
![Page 9: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/9.jpg)
9
![Page 10: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/10.jpg)
10
T cell development can be characterized using flow
cytometry
![Page 11: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/11.jpg)
11
Real data
CD
4
CD8
8 88
13
Double-positives
Double-negatives
CD4 single-positive
CD8 single-positive
![Page 12: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/12.jpg)
12
DN’s can be further subdivided into DN1 through DN4
“Gate” on CD4-CD8-, analyze for CD44 and CD25
CD25 (chain of IL2R)
![Page 13: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/13.jpg)
13
The real data
CD25
(gated on DN cells)
![Page 14: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/14.jpg)
14
Double negative cells appear in the fetal thymus before double positive cells
K. Hogquist
![Page 15: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/15.jpg)
15
T cell populations during development and in the lymphoid tissue
8.383.24.711.47.9
CD4
CD8
THYMUS SPLEEN
![Page 16: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/16.jpg)
16
Thymocytes at different developmental stages are found in distinct parts of the thymus
Maturation
![Page 17: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/17.jpg)
17
Today
Thursday
![Page 18: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/18.jpg)
18
Most cells fail to complete thymocyte maturation
![Page 19: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/19.jpg)
19
macrophagesapoptotic cells
(DNA fragmentation)
Macrophages...garbage collectors, contain the dying cells
Most developing thymocytes die in the thymus
![Page 20: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/20.jpg)
20
T cell development involves the sequential generation, assembly and testing of the newly rearranged TCR
![Page 21: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/21.jpg)
21
TCR rearrangement is very similar to Ig rearrangement
![Page 22: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/22.jpg)
22
Figure 5-6 part 1 of 3
First step: TCRbeta rearrangement
![Page 23: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/23.jpg)
23
Figure 5-6 part 2 of 3
![Page 24: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/24.jpg)
24
Pre-T cell receptor
How do you test for successful TCRbeta chain rearrangements if you have not
rearranged TCRalpha?Pre-Talpha
![Page 25: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/25.jpg)
25
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
![Page 26: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/26.jpg)
26
Remove extracellular domains…
…and examine T cell development
(work of Nigel Killeen, UCSF)
Does the pre-TCR have a ligand?
![Page 27: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/27.jpg)
27
pTT;T receptor restores CD4+8+ development and thymocyte expansion
(Irving (1998) Science 280:905)
Pre-TCR extracellular domains are not required!
![Page 28: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/28.jpg)
28
CD25
Forward scatter(cell size)
Proliferating cells are bigger
-selection leads to proliferation
DN2
DN3
Gated on CD4/CD8 DNs
![Page 29: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/29.jpg)
29
TCR locus has two D-J clusters
Allows a 2nd rearrangement if 1st is nonproductive
![Page 30: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/30.jpg)
30
We are here
![Page 31: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/31.jpg)
31
Progression through development correlates with rearrangement
![Page 32: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/32.jpg)
32
At the DP stage, TCRalpha rearrangement begins
![Page 33: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/33.jpg)
33
TCR locus has 50 J segments
![Page 34: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/34.jpg)
34
TCR locus-- replacement rearrangement
![Page 35: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/35.jpg)
35
DP thymocytes express surface TCR
CD3 lo cell-- pre-TCRCD3 hi cells-- TCR
![Page 36: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/36.jpg)
36
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
![Page 37: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/37.jpg)
37
Differential expression of Src family kinases
![Page 38: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/38.jpg)
38
Remember what Lck does
![Page 39: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/39.jpg)
39
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!
![Page 40: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/40.jpg)
40
Thursday
vs
![Page 41: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/41.jpg)
41
A model for versus lineage commitment
![Page 42: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/42.jpg)
42
TCR rearrangement removes the delta locus
![Page 43: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/43.jpg)
4310%90%
TCR lineage comprises the majority of T cells
![Page 44: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/44.jpg)
44
T cells are favored during early fetal development
![Page 45: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/45.jpg)
45
Waves of T cells with specific TCR usage develop early gestation
![Page 46: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/46.jpg)
46
Waves of T cells home to different tissues
![Page 47: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/47.jpg)
47
• 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
![Page 48: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/48.jpg)
48
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
![Page 49: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/49.jpg)
49
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
![Page 50: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/50.jpg)
50
DETCs promote wound healing
![Page 51: 1 T Cell Development I: The Generation of TCR + Thymocytes.](https://reader035.fdocuments.net/reader035/viewer/2022062516/56649d775503460f94a5a027/html5/thumbnails/51.jpg)
51
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