T-Cell Maturation, Activation, And Differentiation

W. Robert Fleischmann, Ph.D.

Department of Urologic Surgery

University of Minnesota Medical School

rfleisch@umn.edu

(612) 626-5034

Objectives

• To understand T cell maturation in the thymus, including positive and negative selection

• To understand T cell activation, including signals initiated by antigen recognition and by costimulatory signaling

• To understand T cell differentiation and the generation of the various subsets of T cells

T Cell Maturation

T Cell Maturation

• B cells are mature when they leave the bone marrow.

• T cells require “higher education” after they leave the bone marrow in order to mature.– T cell maturation occurs in the thymus.– Maturing T cells are called thymocytes.– Maturing T cells undergo positive and

negative selection.

Key Feature of the Thymus

• Thymic stromal cells express the protein Notch. – In the absence of notch, no T cell

maturation occurs.– If hematopoietic stem cells are transfected

to express notch, then only T cells develop.– Thus, notch is a key to the differentiation of

the immature T cells to mature T cells.

T Cell Maturation in the Thymus

• The T cell precursors enter the thymus as a double negative (CD4-CD8- or DN) cell.

• They begin a process of dividing and differentiating that takes about 3 weeks.– Pass through 4 stages of DN cells – Become double positive (DP) cells – Ultimately become CD4+ or CD8+.– Leave the thymus as mature T cells.

T Cell Maturation

Strategic Steps1. Move from BM to

thymus and express…a. CD44 for thymus

localizationb. c-Kit for replicationc. CD25 (IL-2R) for IL-2-

driven replication

2. TCR Rearrangementsa. chain is first. a chain is second

3. Surface moleculesa. CD4-CD8- initiallyb. CD4+CD8+ after a

TCRc. CD4+CD8- or CD4-

CD8+

T Cell Selection in the Thymus

• Positive Selection = MHC Restriction:– Permits survival of only those T cells that

recognize self-MHC molecules.– Thus, it is responsible for the selection of

the self-MHC-restricted T cell repertoire.

• Negative Selection = Self-Tolerance:– Eliminates T cells that respond too

strongly to self MHC or with self MHC plus self peptides.

– It is responsible for the development of a primary T cell repertoire that is self-tolerant. This process is called the development of Central Tolerance.

Cost of T Cell Maturation

• It is estimated that 98% of thymocytes do not mature into mature T cells.

• Most are eliminated by apoptosis because– They fail to make productive TCR rearrangements– They fail to survive thymic selection

T Cell Activation

Multiple Genes Are Activated by Ag Binding

• Immediate Early Genes– Expressed within 30 minutes of antigen

recognition– Encode a number of transcription factors: c-Fos, c-

Myc, c-Jun, NFAT, and NF-B

• Early Genes– Expressed within 1-2 hrs of antigen recognition– Encode IL-2, IL-2R, IL-3, IL-6, IFN-, other

proteins

• Late Genes– Expressed more than 2 days after antigen

recognition– Encode a number of adhesion molecules

•P56lck phosphorylates ITAMs on chains, creating a docking site for ZAP-70.

•ZAP-70 phosphorylates adaptor molecules that activate other enzymes.

•Phospholipase C

activation causes

breakdown of

phosphoinositol

bisphosphate (PIP2) to•inositol 1,4,5 triphosphate

(IP3)

•diacylglycerol (DAG)

•Inositol 1,4,5 triphosphate (IP3)

–Causes rapid release of Ca++

from endoplasmic reticulum

–Opens Ca++ channels in the

cell membrane

–Activates the transcription

factor NFAT that is required

for transcription of IL-2, IL-4

•Diacylglycerol (DAG)–Activates protein kinase C

which phosphorylates many

targets

–Activates the transcription

factor NF-B that is in turn,

required for transcription of

IL-2

•Guanine nucleotide exchange factor (GEF) induces Ras and Rac pathways that lead to cell division.

•The RAS/MAP Kinase Pathway

–Ras is a small G protein that, when activated by GTP, initiates a cascade of protein kinases called the mitogen activated protein kinase pathway (MAP kinase pathway).–Activation of transcription complex of Fos/Jun/AP-1 that activates a number of genes, including those involved in initiation of cell division.

Sensitivity of TCR:Antigen Binding for T Cell Activation

• Binding of one TCR on a T cell to its cognate antigen is sufficient to trigger the activation of the T cell.

• Incremental T cell activation occurs with more TCR:antigen bindings.

• Maximal T cell activation occurs when 10 TCR:antigen bindings have occurred.

Costimulatory Signals

• Helper T cell activation requires two binding signals.– Signal 1: the initial signal generated

by TCR:antigen recognition– Signal 2: the second signal (non-

specific for antigen) is provided when CD28 on the T cell interacts with B7 on the antigen-presenting cell.

• Helper T cell activation requires cytokine signals.

Costimulatory Signal Regulation

• Resting T cell– Expresses CD28– Activation signal is

transduced when CD28 is bound by B7

– CTLA-4 induced

• Activated T cell– Expresses CTLA-4 in

addition to CD28– Inhibitory signal is

transduced when CTLA-4 is bound by B7, providing a brake on activation and proliferation

Clonal Anergy

• What if there is no costimulatory signal mediated by B7 binding to CD28– The T cell is in a non-responsive state

(clonal anergy).– It cannot respond to the TCR:antigen

binding signal.

Superantigens• Some antigens can bind

both to the MHC and to certain TCR molecules, without residing in the antigen groove of the MHC molecule.– Initiate a non-specific

interaction– Stimulate many T cells of

different antigenic specificities to divide and differentiate

– Called superantigens– Activation of so many T cell

clones can have serious consequences, such as the over induction of IFN- and TNF-a associated with toxic shock.

T Cell Differentiation

The Naïve T Cell Population• T cells leave the thymus as naïve T cells.

– There are about 2X as many CD4+ T cells as CD8+ T cells in the periphery.

– The T cells are in G0, or the resting phase of the cell cycle.

• The naïve T cells constantly circulate from blood, to lymph, to lymphoid tissues, and back to blood in a cycle that takes about 12-24 hrs.– If a naïve T cell encounters its cognate antigen in

the lymph node, it remains there.

• The rapid recirculation of naïve T cells is necessary because only about 1:105 naïve T cells has specificity for any given antigen.

Th Cell Differentiation• Binding of the TCR to its cognate

antigen initiates the primary response.– After about 24 hrs, the responding T

cell enlarges to form a blast cell and begins to undergo rounds of cell division.

– IL-2 synthesis is increased by 100-fold by induction of IL-2 mRNA synthesis and by stabilization of IL-2 mRNA.

– IL-2 binding to the high affinity IL-2 receptor (also induced after antigen binding) activates the proliferation: 2-3 division/day for 4-5 days to generate a clone of responding T cells.

– Some of the responding T cells become effector T cells; others become memory T cells.

T Cell Apoptosis

• After undergoing rapid proliferation, effector T cells must undergo apoptosis or we would become blobs of T cells.– FasL-mediated apoptosis:

• Death in 2-4 hours

– MHC/Ag-mediated apoptosis:

• Death in 8-10 hours

• Note that memory T cells do not undergo apoptosis.

Effector T Cells• Effector T cells can be induced from naïve T

cells or from memory T cells upon exposure to cognate antigen.

• Effector T cells are short-lived, surviving for a few days to a few weeks.

• Effector T cells can be of several types.– CD4+ Helper T cells

• Th1 subset secretes IL-2, IFN-, TNF- and stimulates cell mediated immunity.

• Th2 subset secretes IL-4, IL-5, IL-6, IL-10 and stimulates humoral (antibody mediated) immunity

– CD8+ Cytotoxic T cells

Memory T Cells• Memory T cells can be induced from naïve T cells or

from effector T cells after antigenic activation and differentiation.

• Memory T cells are long-lived, surviving for many years.

• Memory T cells can be reactivated by re-exposure to cognate antigen to become effector cells (secondary response).

• There are no identifying surface markers that can be used to differentiate memory and effector T cells.

• While naïve T cells are almost exclusively activated by dendritic cells, memory T cells can be activated by macrophages, dendritic cells, and B cells (thought to be a function of high levels of adhesion molecules).

Regulatory T Cells• The CD4+CD25+FoxP3+ subpopulation

of T cells can suppress the immune response (regulatory T cells, Treg cells).

• Others cells may also have regulatory activity.

• Loss of Treg cells by Ab depletion has caused development of autoimmunity.

Details of Thymic Maturation Events

Stages of T Cell Maturation

• DN1 cells (c-kit+, CD44high, CD25- cells) enter the thymus.– CD44high is needed for localization to thymus.– c-Kit+ is a receptor for stem cell factor and is

needed for initiation of growth in the thymic environment.

– DN1 cells respond to the thymic environment by beginning to proliferate and to express CD25 (IL-2R).

– DN1 cells are capable of giving rise to all subsets of T cells.

Stages of T Cell Maturation

• DN2 cells have turned on synthesis of CD25 (c-kit+, CD44low, CD25+ cells).– They turn on RAG-1 and RAG-2 and begin

rearranging TCR , , and .– TCR a does not begin rearrangement

because its DNA region is too condensed.

• Cells destined to express TCR diverge from the other T cells with the transition from DN2 to DN3 and leave the thymus.

Stages of T Cell Maturation

• DN3 cells have turned off c-kit and CD44 (c-kit-, CD44-, CD25+ cells).– The DN3 cells halt their proliferation.– TCR is rearranged.– It combines with a 33 kDa protein known

as the pre-Ta chain.– This dimer associates with the CD3 group

of molecules to form a complex called the pre-T cell receptor or the pre-TCR.

T Cells Bearing the Pre-TCR

• Once the Pre-TCR is produced, an activation signal can be transduced across the membrane to initiate several actions.– Indicates that the cell has made a TCR chain

and signals further proliferation and maturation.– Suppresses further rearrangement of TCR ,

resulting in allelic exclusion.– Permits the cell to rearrange the TCR a chain.– Induces developmental progression to the

CD4+CD8+ double-positive T cell.• Delayed synthesis of TCR a chain gives a

tremendous increase in the diversity of the T cells, since each T cell with a given chain can express a different a chain.

Stages of T Cell Maturation

• DN4 cells turn off expression of CD25 (c-kit-, CD44-, CD25- cells).– Expression of CD4 and CD8 is turned on.

• Double positive T cells (CD4+CD8+ T cells) – Rapid proliferation occurs, creating a clone of cells with the

same TCR chain.– After a period of time, proliferation stops and the TCR a

chain is sythesized. – Delayed synthesis of TCR a chain gives a tremendous

increase in the diversity of the T cells, since each T cell with a given chain can express a different a chain.

– Expression of a functional TCR permits the T cell to undergo positive and negative selection.

Stages of T Cell Maturation

• Double positive T cells (CD4+CD8+ T cells) lose one of their T cell markers and become CD4+ or CD8+ T cells.

• The single-positive T cells undergo additional negative selection.