Development

T lymphocyte Development

Where do T cells come from?

1. Multipotent Lymphoid Progenitors Migrate from the Bone Marrow to the Thymus

2. In the Thymus, the Lymphoid Progenitors Differentiate to pre-T Cells and are Educated to Differentiate Self from Non-self

3. Positively Selected T Cells Emigrate from the Thymus to Mediate and Effect the Cognate Immune Response

T Lymphocyte Maturation in the Thymus

The CD4 by CD8 FACS Plot as an Indicator of Normal (and Abnormal)

Thymocyte Development

CD4

CD8

Ikaros controls commitment to Lymphoid lineage

(Ikarus DN)

Bone Marrow

Thymus

Notch 1 is required for T cell lineage commitment

Control Notch1-/-

CD4

Ratke et al, Immunity, 1999

How Notch Signaling Works

Ligands expressedIn the thymus

Reciprocal Regulation

Pax-5 induces the expressionOf B lineage genes (CD19, BLNK)And represses the expression of Notch

But really location controls the T cell versus B cell Lineage Choice

So what controls homing of CLPs to the thymus?

And what controls the expression of these homing receptors?

T Lymphocyte Maturation in the Thymus

As T Lymphocytes Develop, They Migrate From the Thymic

M/C junction to the Cortex

to the Medulla

DN1 Cells in the Thymus Are Not Committed to become T Lymphocytes

• TCR loci are in germline configuration

• Cell can differentiate to become a B lymphocyte, Natural Killer cell, or Dendritic cell (Michie et al, JI, 2000; Ikawa et al, JEM, 1999;Sanchez et al, JEM, 1994)

The DN2 Subset of Thymocytes are More Committed, but Not Quite Exclusive

• TCR loci are in germline configuration

• DN2 thymocytes may give rise to dendritic cell(Moore et al, 1995; Wu et al 1996; Ardavin et al, Nature 1993; Shortman et al, Imm Rev. 1998)

• DN2 thymocytes no longer differentiate to the NK cell or B lymphocyte lineages

DN3 Cells are Committed to the T Lineage

• Downregulation of CD44 expression

• Upregulation of RAG genes

• V-D-J recombination of TCR chain locus

• Expression of pre-TCR chain

The pre-TCR chain (DN3)

• Invariant Type I TM protein

• Contains only one Ig Domain

• Physically associated with TCR chain

• Signaling appears to be ligand-independent– TCR chain in this complex

lacking extracellular domain is sufficient to allow progression to DP stage

From Fundamental Immunology, 4th ed. (Paul)

Targeted Gene Mutants Unable to Mature to the DP Stage

After Assembly of a Functional pre-TCR

• Shut down of TCR rearrangement; TCR allelic exclusion

• Onset of proliferation/expansion

• Differentiation to DN4, CD8ISP, and then DP

Three Models of vs. Lineage Determination

• Earliest (fetal) waves of T cells are mostly

• Later production is skewed toward lineage

• Conflicting Data Supporting Each Model

• Mature T cells have rearranged locus; ’s have rearranged locus

• Even DN4 cells (which have selected a chain) can become lymphocytes in transfer experiments

From Fundamental Immunology, 4th ed. (Paul)

WntTcf/Lef

Notch

IL-7Jak3/

BMPSmad

SCFc-kitCXCR4

V(D)J Recombination in developing T cells

• chain occurs first (DN3 stage) – VDJ• chain occurs second – VJ, no D region• Allelic exclusion• Combinatory diversity of the adaptive immune system

• This is a big problem

Fates Awaiting a DP Thymocyte

(95%)

Default

Positive Selection: Host MHC determines CTL immune responsiveness (Bevan, Nature, 1977)

d(with peptides) b(with peptides) B10(H-2d) B10(H-2b)H-2d/b BM -> H-2b/d +++ +++H-2d/b BM -> H-2d +++ +H-2d/b BM -> H-2b + +++

Balb/c H-2d/b BM -> H-2b/d Balb/c H-2d/b BM -> H-2d Balb/c H-2d/b BM -> H-2b

immunize with B10H-2d/b splenocytesrestim in vitro look for MHC restricted cytotoxicity

Evidence for MHC Restriction of CD4+ and CD8+ T Lymphocytes is Apparent in MHC KO Mice

CD4

CD8

(Grusby et al, PNAS, 1993)

Negative SelectionHY TCR Tg is negatively selected by a male specific

antigen

HY

TC

R fe

mal

eH

Y T

CR

Mal

e

(Kisielow et al, Nature, 1988)

Negative and Positively selecting peptides different in there Kd

(Alam et al, Nature, 1996)

Model of Thymocyte Education

Signal Transduction in thymocyte development

TCR Signal Transduction

But what are the down stream effectors?

Which downstream pathways contribute to thymocyte selection?

Do Positive and Negative Selection Signals Activate the Same Signal Transduction Pathways?

Disruption of the ERK Pathway Interferes With Positive but not Negative Selection (an effect on

differentiation and proliferation?

• Also:– DN Mek1

• Alberola-Ila et al, 1995• Alberola-Ila et al, 1996

– DN p21ras

• Swan et al, 1995• Alberola-Ila et al, 1996

– DN Raf• O’Shea et al, 1996

– RasGRP• Dower et al, 2000

– Indirect (ERK Induction from CD3 chain)

• Werlen et al, 2000• Delgado et al, 2000

ERK1 ERK1

Pages et al 1999 Science 286:1376

Calcineurin B is required for positive selection

Neilson et al, 2004

Cnb1deficient and Control Thymocytes are Equally Sensitive to TCR Crosslinking and

Peptide Complexed with MHC

MCC-Mediated Cell Death dose response

-12 -11 -10 -9 -8 -7 -6 -5 -40

20

40

60

80

100Cnbf/

Lck-Cre+ Cnbf/

concentration MCC peptide [2 X 10 X M]

%P

I-A

V-D

P t

hym

ocy

tes

rela

tive

to

co

ntr

ol

Neilson et al, 2004

Via

bili

ty

Which downstream pathways contribute to thymocyte selection?

(positive selection)(positive selection)

Vav GEF is required for positive selection

Active Rac1 changes positive to negative selection

Female Female/active Rac1

(Gomez et al, Immunity, 2001)

Data = Explanation?• Synapse during negative selection (Richie et al, Immunity, 2002)

• Synapse formation in peripheral T cells requires Vav/actin dynamics (Holsinger et al, Curr. Bio, 1998; Wulfing et al, PNAS, 2000)

• Positive selection likely does not form a synapse (Peter

Ebert unpublished)

• Therefore, lacking Vav/Rac etc. changes negative to positive selection via an effect on synapse formation and subsequent signaling

Which downstream pathways contribute to thymocyte selection?

(positive selection)(positive selection) (Modulator

Both)

Negative Selection is Impaired in DP Thymocytes Deficient in the Pro-Apoptotic Bcl-2 Family Member Bim

Bouillet et al 2002 Nature 618:108

Three Models for the CD4 vs CD8 Choice

From Fundamental Immunology, 4th ed. (Paul)

Modulation of lck activity can alter CD4/8 lineage commitment (based on the fact that the cytoplasmic tail of CD4 binds to

more lck than CD8)

Dn-lck Act-lck

Other studies indicate that the duration of signal controls CD4 versus CD8 with CD4 requiring longer(Yasutomo et al, Nature, 2000)

Regulatory T cells may develop in the Thymus

HA specific TCR Transgenic mouseMouse expressing HA peptide

TCR Tg CD25

2D model

Hypothetical 3D model of thymocyte development

What a DP Thymocyte Needs to Progress to the SP Stage

• Everything it Needed to Become DP

• Functional TCR chain rearrangement

• CD4 and MHC II (To be a CD4+ cell)

• CD8, MHC I and TAP (To be a CD8+ cell)

• ERK signaling

• Calcineurin signaling

T Lymphocyte Maturation in the Thymus

T Lymphocyte Maturation after leaving the Thymus

Some evidence that cells that haverecently left the thymus may have special “status” in the periphery in regards to tolerance induction or the ability to homeostatically proliferate

Antigen presentation and MHC expression control thymocyte selection

•DC•Medullary Epithelial cells•Cortical Epithelial cells•Endothelial cells

DO CERTAIN CELL TYPE CONTROL DIFFERENT T CELL DEVELOMENTAL FATES?-Positive selection-Negative selection-Receptor editing

Aire-/- mice develop autoimmunity because Aire Mediates

Ectopic Gene Expression in Medullary Stroma

Ohashi 2002 Science, 298:1348

The Transcription Factor Autoimmune Regulator (Aire) Mediates Ectopic Gene

Expression in the Thymic Medullary Stroma

Ohashi 2002 Science, 298:1348

Take Home Messages• Notch steers CLP’s to T lymphocyte Fate• TCR chains undergo V(D)J recombination to generate

diversity; they also exhibit allelic exclusion– TCR chain is selected with an invariant pT chain at the DN3

stage– TCR chain is selected with pre-existing TCR chain at the DP

stage

• DP Thymocytes Undergo Positive and Negative Selection to Generate a Population of Mature T Lymphocytes that can Recognize Self MHC with Intermediate Affinity

• Recent Evidence Indicates that Positive and Negative Selection Are Mediated by Distinct Pathways

• APC in the thymus may present self antigen that effect negative selection

T Lymphocyte Development and Function is Controlled at Various Stages of Development

by Signals through the T Cell Receptor

Signaling 1983