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 ControlNotch1-/- CD4 Ratke et al, Immunity, 1999

How Notch Signaling Works Ligands expressed In the thymus

Reciprocal Regulation Pax-5 induces the expression Of 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, 4 th 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, 4 th ed. (Paul)

Wnt Tcf/Lef Notch IL-7 Jak3/ BMP Smad SCF c-kit CXCR4

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 splenocytes restim 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 Selection HY TCR Tg is negatively selected by a male specific antigen HY TCR female HY TCR Male (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 p21 ras 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 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 Neilson et al, 2004 Viability

Which downstream pathways contribute to thymocyte selection? (positive selection)

Vav GEF is required for positive selection

Active Rac1 changes positive to negative selection FemaleFemale/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) (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, 4 th 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 mouse Mouse expressing HA peptide TCR TgCD25

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 have recently 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