Transcript regulatory T cells, Treg cells

T-Cell Maturation, Activation, And
Differentiation
W. Robert Fleischmann, Ph.D.
Department of Urologic Surgery
University of Minnesota Medical School
[email protected]
(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
James Cormack, age 22 months, is brought to your
clinic by State authorities. He was picked up from a
day-care center following a report of possible child
abuse. The examining physician notes that James has
multiple severe bruises on his body.
James’s family has not yet been contacted by the
State juvenile authorities, so no medical history is
available.
The physician notes that James has a stuffy nose.
An examination indicates that James has a purulent
discharge from his nose, consistent with sinusitis.
James also appears to have eczema on his trunk.
The physician requests a complete WBC and sends
a sample of the discharge for culture.
Complete WBC results:
Total WBC counts:
Differential WBC counts*:
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
12,000/µl
(4,100-10,900/µl)
35%
60%
5%
0%
0%
(22-40%)
(49-73%)
(3-10%)
(0-6%)
(0-3%)
23 sec
13 sec
30,000/µl
(18-28 sec)
(12-14 sec)
(140,000-450,000/µl)
RBC parameters: all normal
Partial thromoplastin time
Prothrombin time
Platelet count
What do these results suggest?
*Values for a patient who is 2 weeks to 4 years old.
Using the telephone number provided by the day-care
center, James’s mother is contacted.
She arrives at the hospital and is appalled and upset to
learn that James had been taken into custody by State
authorities and transported to the hospital without her and
her husband’s knowledge.
Once she is sufficiently calm, she provides the physician
with James’s social and medical history.
She indicates that she and James have moved to the
U.S. from Ireland two weeks ago, catching up with her
husband who began a post-doctoral fellowship at the local
university a month earlier. She dropped James off at a
local church’s day-care center and went job hunting.
She indicates that James has had a history of bruising
and says that it seems to be getting worse lately.
However, she thinks his bruising is due to the combination
of his greater level of activity and increasingly risky
behavior (climbing on things and running out of control into
furniture).
When asked, she also mentions that James has a
number of ear and sinus infections. Several months ago,
he was diagnosed with pneumonia. She doesn’t know
what bacteria have been responsible for these infections.
In response to a specific question, she indicates that
James has been staying at home with her or with his
grandparents. He has not been at a day care center.
What do these results suggest?
What would you do next?
Number of B cells and T cells in peripheral blood:
T cells
80%
CD4+
54%
CD8+
26%
NK cells
10%
B cells
10%
(60-95%)
(60-75%)
(25-30%)
(4-30%)
(4-25%)
Response to T cell mitogens:
Concanavalin A: normal response
Phytohemagglutinin: normal response
Candida albicans: no response
Response to B cell mitogen
Pokeweed mitogen: normal response
Immunoglobulin levels in blood:
Serum IgG
350 mg/100 ml
Serum IgA
40 mg/100 ml
Serum IgM
13 mg/100 ml
What do these results suggest?
350-800 mg/100 ml
15-50 mg/100 ml
25-75 mg/100 ml
Culture results from nasal swab: Streptococcus pneumoniae
Mean volume of platelets: <5 fL
What do these results suggest?
(7.5-11.5 fL)
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
1.
Strategic Steps
Move from BM to
thymus and express…
a.
b.
c.
2.
TCR Rearrangements
a.
.
3.
CD44 for thymus
localization
c-Kit for replication
CD25 (IL-2R) for IL-2driven replication
 chain is first
a chain is second
Surface molecules
a.
b.
c.
CD4-CD8- initially
CD4+CD8+ after a
TCR
CD4+CD8- or CD4CD8+
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 selftolerant. 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, cMyc, 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 (nonspecific 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 1224 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.
James Cormack
Because of the lack of T cell responsiveness
to Candida albicans and low IgM levels, the
physician suspects a T cell disorder.
The physician knows (from checking
uptodate.com) that among the potential causes
of thrombocytemia, Wiskott-Aldrich syndrome
stands out as being the only one that is an
immunodeficiency condition.
The physician contacts the Immune Deficiency
Foundation and sends sample for genetic testing
for suspected Wiskott-Aldrich syndrome. The
results of the genetic testing confirm that James
has Wiskott-Aldrich syndrome.
James Cormack
Patients with Wiscott-Aldrich syndrome have a
mutation in the Wiscott-Aldrich protein (WASp).
Individuals with mutant WASp have defects in
IL-2 production, resulting in impaired T cell
proliferation. Thus, individuals with mutant
WASp protein is have impaired helper T cell
induction of antibody production and impaired
helper T cell induction of CD8+ T cell function.
WASp is also required for platelet production.
In the absence of platelets, bruising easily occurs
The physician arranges for James to have a
bone marrow transplant.
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 (IL2R).
– 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.
Key Players in Activation
• 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.