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.human anatomyanteriorchest cavity 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, theapoptotic thymus was originally dismissed as a "lymphocyte graveyard", without functional importance. The importance of the thymus in the immune system wasimmune system discovered in 1961 by Jacques Miller, by surgicallyJacques Miller 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.immunology

Function 1.In the two thymic lobes, lymphocyte precursors from the bone- marrow become thymocytes, and subsequently mature into T cells.lymphocytethymocytes 2.The ability of T cells to recognize foreign antigens is mediated by the T cell receptor.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.T cell receptor thymocytepeptideMHC 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.central toleranceT cell receptor 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.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 of numerous lobules held together bylobules delicate areolar tissue; the entire organareolar tissue being enclosed in an investing capsulecapsule of a similar but denser structure. The primary lobules vary in size from that of a pin's head to that of a small pea, and are made up of a number of small nodules ornodules folliclesfollicles.The follicles are irregular in shape and are more or less fused together, especially toward the interior of the organ. Each follicle is from 1 to 2follicle mm in diameter and consists of a medullarymedullary and a cortical portion,cortical and these differ in many essential particulars 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 CD44 CD25

Positive vs. Negative Selection

Shortman CD4 low CD117 + CD44 + CD25 - THYMUSTHYMUS SCZ Medulla CMJ DN4 CD44 - CD25 - Inner Cortex Outer Cortex CD4 + SP Late Medullary (CD69 - CD62L + ) CD4 + SP Early Medullary (CD69 + CD62L - ) CD8 + SP DN2 CD44 + CD25 + DP CD4 + CD8 + DN3 CD44 - CD25 + Periphery

Compartmentalization?

CCR9 / CCL25 CCL25 CC Chemokine (a.k.a TECK) Sole known ligand for CCR9 CCR9 CC Chemokine Receptor 7 transmembrane domain G protein- coupled receptor Sole known receptor for CCL25

CCR9 / CCL25 Highly expressed in both human and mouse Thymus (Thymus = Cortex & Medulla) CCR9 expressed by developing Thymocytes (Cortex>Medulla) CCL25 expressed 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 DNDP CD4+CD8+ mCCR9 Actin mCCR9 Actin no cDNA RT+RT-RT+RT- RT+RT-RT+RT- % mCCR9 Expression Relative to that in DP Cells AB

CCR9 / CCL25 Useful Tools: Generation of CCR9-deficient mice CCL25-deficient mice Generation of mouse anti-mouse CCR9 mAbs WTCCR9 -/- Whole Thymocytes CCR9 (clone CW-1.2 IgG2a) mouse IgG2a Isotype CTRL

Wild-typeCCR9 -/ CD4 CD8 CD3 81.4% 12.1% 76.6% 9.5%

WTCCR9 -/- Expression of CCR9 by Early Thymic Subsets DN2 DN3 DN4 DP Shortman

CD8 + SP CD4 + SP DP WTCCR9 -/- CCR9 is highly expressed by WT DP, down regulated by WT CD4 + SP and maintained by WT CD8 + SP

Shortman CD4 low CD117 + CD44 + CD25 - DN2 CD44 + CD25 + DN3 CD44 - CD25 + DN4 CD44 - CD25 - DN4’ Escape From SCZ? DP CD4 + CD8 + CD4 + SP Early Medullary (CD69 + CD62L - ) CD8 + SP CCR9 neg CCR9 low CCR9 int CCR9 high CCR9 low/neg CCR9 high/int THYMUSTHYMUS CD4 + SP Late Medullary (CD69 - CD62L + ) SCZ Medulla CMJ Inner Cortex Outer Cortex Periphery ?

Other chemokine/receptor pairs? Pro-T DN DP SP CD4 + SP CD8 + CXCR4/CXCL12 CCR9/CCL25 CCR7/CCL21 XCL1/XCR1 NK TCR  CCR7/CCL19,CCL21 CCR4/CCL22,CCL17 CCR6/CCL20 CCR8/CCL1 ? CCR5/CCL4 ? CCR3/CCL11 thymus CCR7/CCL19,CCL21 CCR9/CCL25 CCR3/CCL11 CXCR3/CXCL ? CXCR4/CXCL12 CCR8/CCL1 sélection par le stroma thymique CCR9/CCL25 CXCR4/CXCL12 CCR5/CCL4, CCL5 CXCR3/CXCL CCR8/CCL1 cortex sous-capsulaire et superficiel cortex profond - jonction cortico-médullaire - médulla ? CXCR4/CXCL12 CCR9/CCL25 CCR4/CCL22,CCL17 CCR3/CCL11 CCR7/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 Jul;86(13):5094-8