thymic T cell differentiation and compartmentalization

Marc-André Wurbel, Ph.D.

In human anatomy, the thymus is an organ located in the upper anterior portion of the chest cavity just behind the sternum.

It is of central importance in the maturation of T lymphocytes.

The thymus is of a pinkish-gray color, soft, and lobulated on its surfaces. At birth it is about 5 cm in length, 4 cm in breadth, and about 6 mm in thickness. The organ enlarges during childhood, and atrophies at puberty.

History

• Due to the large numbers of apoptotic lymphocytes, the

thymus was originally dismissed as a "lymphocyte

graveyard", without functional importance.• The importance of the thymus in the immune system was

discovered in 1961 by Jacques Miller, by surgically

removing the thymus from three day old mice, and

observing the subsequent deficiency in a lymphocyte population,

subsequently named T cells after the organ of their origin.• Recently advances in immunology have allowed the fine

dissection of the function of the thymus in T cell maturation.

Function

1. In the two thymic lobes, lymphocyte precursors from the bone-marrow become thymocytes, and subsequently mature into T cells.

2. The ability of T cells to recognize foreign antigens is mediated by the T cell receptor.

3. The T cell receptor undergoes genetic rearrangement during thymocyte maturation, resulting in each T cell bearing a unique T cell receptor, specific to a limited set of peptide:MHC combinations.

4. The random nature of the genetic rearrangement results in a requirement of central tolerance mechanisms to remove or inactivate those T cells which bear a T cell receptor with the ability to recognize self-peptides.

5. Once mature, T cells emigrate from the thymus and constitute the peripheral T cell repertoire responsible for directing many facets of the adaptive immune system.

6. Loss of the thymus at an early age through genetic mutation or surgical removal results in severe immunodeficiency and a high susceptibility to infection.

1- Structure

Each lateral lobe is composed ofnumerous lobules held together bydelicate areolar tissue; the entire organbeing enclosed in an investingcapsule of a similar but denserstructure. The primary lobules vary insize from that of a pin's head to that ofa small pea, and are made up of anumber of small nodules orfollicles.The follicles are irregular inshape and are more or less fusedtogether, especially toward the interiorof the organ. Each follicle is from 1 to 2mm in diameter and consists of amedullary and a cortical portion,and these differ in many essentialparticulars from each other.

2- T Cell Receptor

• TCR and chains

• (TCR and chains)

• CD3 units

3- TCR genes and their rearrangements

4- T cell differentiation

TCR rearrangement

TCR rearrangement

CD

44

CD25

Positive vs. Negative Selection

ShortmanCD4lowCD117+CD44+CD25-

THYMUS

SCZ

Medulla

CMJ

DN4CD44-CD25-

InnerCortex

OuterCortex

CD4+ SPLate Medullary (CD69-CD62L+)

CD4+ SPEarly Medullary (CD69+CD62L-)

CD8+ SP

DN2CD44+CD25+

DPCD4+CD8+

DN3CD44-CD25+

Periphery

Compartmentalization?

CCR9 / CCL25

• CCL25CC Chemokine (a.k.a TECK)Sole known ligand for CCR9

• CCR9CC Chemokine Receptor7 transmembrane domain G protein- coupled receptorSole known receptor for CCL25

CCR9 / CCL25

• Highly expressed in both human and mouse Thymus(Thymus = Cortex & Medulla)

• CCR9expressed by developing Thymocytes (Cortex>Medulla)

• CCL25expressed in the by all thymic cortical epithelial cell and some scattered epithelial cells in the medulla

Semi-quantitative RT-PCR : CCR9 expression analysis within

thymic subsets

DN DP

CD4+ CD8+

mCCR9Actin

mCCR9Actin

no c

DN

An

o c

DN

A

RT+ RT- RT+ RT-

RT+ RT- RT+ RT-

8.7 %

16.5 %

10.9 %

100 %

0

10

20

30

40

50

60

70

80

90

100

DN DP CD4 CD8

% m

CC

R9 E

xp

ressio

n R

ela

tive t

o t

hat

in D

P C

ells

A B

CCR9 / CCL25Useful Tools:

• Generation ofCCR9-deficient miceCCL25-deficient mice

• Generation ofmouse anti-mouse CCR9 mAbs

WT CCR9-/-

WholeThymocytes

CCR9 (clone CW-1.2 IgG2a)mouse IgG2a Isotype CTRL

Wild-type CCR9-/-

2.8

3.2

85.3

8.5

2.2

86.5

8.3

2.9

CD4

CD8

CD3

81.4%

12.1%

76.6%

9.5%

WT CCR9-/-

Expression of CCR9 by Early Thymic Subsets

DN2

DN3

DN4

DP

Shortman

CD8+ SP

CD4+ SP

DP

WT CCR9-/-

CCR9 is highly expressed byWT DP, down regulated by WT CD4+SP and

maintained by WT CD8+ SP

ShortmanCD4lowCD117+CD44+CD25-

DN2CD44+CD25+

DN3CD44-CD25+

DN4CD44-CD25-

DN4’Escape

From SCZ?

DPCD4+CD8+

CD4+ SPEarly Medullary (CD69+CD62L-)

CD8+ SP

CCR9neg

CCR9neg

CCR9low

CCR9low

CCR9int

CCR9high

CCR9high

CCR9low/neg CCR9high/int

THYMUS

CD4+ SPLate Medullary (CD69-CD62L+)

SCZ

Medulla

CMJ

InnerCortex

OuterCortex

Periphery ?

Other chemokine/receptor pairs?

Pro-TDN DP

SP CD4+

SP CD8+

SP CD8+

CXCR4/CXCL12CCR9/CCL25CCR7/CCL21

XCL1/XCR1

NK

TCR

CCR7/CCL19,CCL21CCR4/CCL22,CCL17

CCR6/CCL20CCR8/CCL1

? CCR5/CCL4? CCR3/CCL11

thymus

CCR7/CCL19,CCL21CCR9/CCL25CCR3/CCL11

CXCR3/CXCL9-10-11 CXCR3/CXCL9-10-11

?

CXCR4/CXCL12CCR8/CCL1

sélection par le stroma thymique

CCR9/CCL25CXCR4/CXCL12

CCR5/CCL4, CCL5CXCR3/CXCL9-10-11

CCR8/CCL1

cortex sous-capsulaireet superficiel

cortex profond - jonction cortico-médullaire - médulla

?CXCR4/CXCL12

CCR9/CCL25CCR4/CCL22,CCL17

CCR3/CCL11CCR7/CCL19,CCL21

CCR8/CCL1

périphérie

Other Thymocyte Subsets?

Itohara S, Nakanishi N, Kanagawa O, Kubo R, Tonegawa S. Monoclonal antibodies specific to native murine T-cell receptor gamma delta: analysis of gamma delta T cells during thymic ontogeny and in peripheral lymphoid organs. Proc Natl Acad Sci U S A. 1989 Jul;86(13):5094-8

44+

25-44+

25+44- /low

25+44-/low

25-

CD4+

CD8+

CD8+CD4+

CD8+ Early

CD4+ CD8+

Late

NK

Thymic Dendritic

Cells

CD4+

NK

Thymus Periphery

CD4low ISP DP SPTN

preTCR (TCR DEPENDENT SELECTION

COLONIZATION

NK / T / DC COMMITMENT

COMMITMENT

CD4/CD8 COMMITMENT

EXPORT

TCR DEPENDENT SELECTION

c - kit

IL-7 receptor

pre - TCR

TCR

TCR "TICKLING"

mature

naive

CD8+naive

Bone Marrow

HSC CLP

B

NK TNK T

c (IL-15 ?)

CD1

IL-7R , TCR

TCR rearrangement TCR rearrangement