T Cell Receptor

1
T Cell Receptor T Cell Development
2

• Questions for the next 2 lectures
• How do you generate a diverse T cell population
  
• with functional TCR rearrangements?
• How do you generate a T cell population that is
• self-MHC restricted?
• How do you ensure that those diverse T cell
• receptors are not-self reactive?
• How do you coordinate lineage specification with
  
• MHC specificity and coreceptor expression?
• - ?? vs. ?? T cell
• - CD4 vs. CD8

3
T lymphocyte a key regulator of the immune
system
4
T Lymphocytes

• Arise from stem cells resident in the bone marrow
  and migrate to the thymus which serves as an
  indoctrination center where thymocytes must learn
  to distinguish self from nonself
• Thymocytes that cannot make this induction are
  eliminated, those that can may further
  differentiate, mature, and graduate as T
  lymphocytes and enter the circulation

5
Origin, generation and differentiation of T cells
6
T Cell Development

• T cell progenitors migrate from bone marrow and
  seed thymus.
• T cell progenitors undergo differentiation to
  CD4, CD8 and NKT cells in thymus.
• Mature CD4 and CD8 T cells circulate between
  blood and lymphoid tissues until they meet
  antigens presented on dendritic cells in lymphoid
  tissues.
• T cells further undergo maturation to become
  functional memory or effector T cells in LT

7
Figure 5-2
Thymic involution Human thymus is fully
developed before birth and increases in size
until puberty. It then progressively shrinks
during adult life. Most thymectomized adults
have no problem in T cell immunity because they
have enough memory T cells in the periphery, and
these T cells are long-lived.
8
T Cell Functions

• Collectively, T cells display a number of diverse
  functions
• - They often function to initiate, regulate,
  and
• fine-tune humoral immune response
• - They are effector cells responsible for
  various
• types of cell mediated immune responses
• like DTH, contact sensitivity,
  transplantation
• immunity, and cytotoxicity

9
T Cell Surface Molecules

• TCR A very diverse heterodimer that lacks a
  cytoplasmic tail that would allow direct
  cytoplasmic signaling once TCR binds an epitope
• CD3 Complex It is composed of a group of six
  invariant accessory molecules one CD3 ?, one CD3
  ?, two CD3e, and an intracytoplasmic homodimer of
  ? or CD247 chains
• Cytoplasmic signaling occurs through CD3 that
  noncovalently associate with TCR

10
Figure 3-6
TCR Complex
11

• The CD3 complex is essential for both cell
  surface expression of the TCR and for signal
  transduction once the TCR recognizes an antigen
• Unlike antibodies that can readily bind free
  antigen, a TCR cannot bind soluble antigens, but
  only enzymatically cleaved fragments of larger
  peptides presented as peptide MHC (pMHC)
  complexes

12

• CD4 or CD8 Most mature T cells express CD4 or
  CD8 molecules, they function as important
  co-receptors in association with the TCR
• By binding to invariant portions of the MHC I
  (CD8) or MHC II (CD4), they serve to increase the
  interaction of the MHC-bound antigenic fragment
  and the TCR

13
Figure 3-10
The structures of CD4 and CD8
14
CD8 binds MHC class ICD4 binds MHC class II
Most mature T cells are either CD4 or CD8. CD8
T cells kill cells infected with intracellular
pathogens or tumor cells while CD4 T cells
regulate (activate or suppress) other immune
cells function.
15
TCR Vs Immunoglobulin

• Both
• Bind antigen
• Have Variable region and Constant region
• Have a binding site that is a heterodimer
  (composed of 2 different chains)
• TCRs act only as receptors
• Igs act as receptors and effector molecules
• (soluble antigen-binding molecules)

16
Similarity between TCR and Ig
17
TCR Structure

• The TCR is a member of the immunoglobulin
  supergene family and is composed of two
  polypeptide chains a light a or ? chain and a
  heavy ß or ? chain
• Each polypeptide chain of the heterodimer pair
  contains a variable and a constant region domain
• The Va and V? regions are encoded by V and J gene
  clusters
• The Vß and V? regions are encoded by V, D, and J
  gene clusters
• The D gene cluster provides an additional source
  of variation

18
Figure 3-7
19
a and b TCR gene loci (germline configuration)
20

• The gene clusters undergo DNA rearrangement,
  similar to that already described for
  immunoglobulin genes, to synthesize aß dimers or
  ? ? dimers
• As with immunoglobulins, the constant domain of
  the a and ß or ? and ? chains are encode by
  constant region genes (Ca and Cß or C ? or C ?)
• T cell receptors do not undergo any subsequent
  changes equivalent to isotype switch, and somatic
  hypermutation, important to generating diversity
  of immunoglobulins.

21

• As might be imagined, in the random process of
  generating diversity, a variety of TCR
  specificities would be generated for peptides
  that one may never encounter during his lifetime
• Three distinct categories of TCR specificities
  can be identified
• - Those that recognize peptides that will never
  be
• encountered
• - Those that recognize peptides produced by
  potential
• pathogens or peptides of foreign origin
• - Those that recognize peptides that are
  produced by
• cells of self

22
TcR a gene rearrangement by SOMATIC RECOMBINATION
Spliced TcR ??mRNA
Rearrangement very similar to the IgL chains
23
TcR a gene rearrangement RESCUE PATHWAY
There is only a 13 chance of the join between
the V and J region being in frame

• chain tries for a second time to make a
  productive
• join using new V and J elements

24
TcR b gene rearrangement SOMATIC RECOMBINATION
D-J Joining
V-DJ joining
Rearranged TcR ??1 transcript
C-VDJ joining
Spliced TcR ??mRNA
25
TcR b gene rearrangement RESCUE PATHWAY
There is a 13 chance of productive D-J
rearrangement and a 13 chance of productive V
D-J rearrangement (i.e only a 19 chance of a
productive b chain rearrangement)
Use (DJC)b2 elements
26
TCR gene rearrangements occur in the thymus
The same RSS and the same enzymes are used to
rearrange both the TCR genes and the Ig genes. P
and N nucleotides are added at the junctions
between rearranged segments
n70-80
n52
27
TCR b Chain D region can be read in all frames
J starts with ATTNo additions J is in frame
28
?-chain locus is first to be rearranged
29
Two chances for productive (correct reading
frame) rearrangement b chain
30
?-chain rearrangement
31
Multiple rounds of ?-chain rearrangement can
rescue nonproductive TCR
32
TCR gene rearrangement generates the TCR
repertoire
Pre-TCR complex stops further gene rearrangement
at b locus, and induces thymocyte
proliferation Finally TCR DP cells are made
33
Germline configuration of g and d loci
TCRd D, J and C exons are encoded in the intron
between the the Vs and the Js of the TCRa locus.
The V segments for TCRd (4 known) are mixed in
with the V segments of the TCRa
34
(No Transcript)
35
Figure 3-8 part 2 of 2
Most gd T cells do not express CD4 or CD8. They
are thought to be First line of defense? Bridge
between innate and adaptive responses?
36
Signals through the ??TCR and the pre-TCR compete
to determine thymocyte lineage
37
Generation of diversity in the TcR
COMBINATORIAL DIVERSITY Multiple germline
segments In the human TcR Variable (V) segments
70?, 52? Diversity (D) segments 0?, 2? Joining
(J) segments 61?, 13? The need to pair ? and ?
chains to form a binding site doubles the
potential for diversity JUNCTIONAL
DIVERSITY Addition of non-template encoded (N)
and palindromic (P) nucleotides at imprecise
joints made between V-D-J elements SOMATIC
MUTATION IS NOT USED TO GENERATE DIVERSITY IN TcR
38
The Generation of Diversity (GOD)
39
T Cell Development

• T cell precursors migrate from the bone marrow to
  enter the thymus as thymocytes, they express
  neither aßTCR nor CD4 or CD8 and are called
  double negative (DN) cells
• DN cells proliferate in the subcapsular region of
  the thymus and differentiate to express low
  levels of newly generated aßTCR, both CD4 and
  CD8, and are called double positive (DP) cells

40

• DP cells move inward to the deeper portion of the
  thymus, where they are fated to die within 3-4
  days, unless their TCRs recognize an MHC class I
  or class II molecules on thymic dendritic cells.
  This process is called positive selection
• Although the mechanism of positive selection is
  yet unclear, partial recognition of class II by
  CD4 or class I by CD8 molecules must occur
• T cells that recognize self MHC molecules survive

41

• A DP thymocyte with a TCR that engage MHC class I
  may become a CD8 T cell and a DP thymocyte that
  recognizes MHC class II may become a CD4 T cell
• Class I and class II molecules are not displayed
  on cell surface unless they are loaded with a
  peptide
• Only molecules of self origin are available on
  thymic APCs, and these are presented to the DP
  thymocyte in the deep or medullary area of the
  thymus

42
CD4CD8 DP cells To be CD4 or CD8?
43

• Thymocytes that show strong interaction with MHC
  molecules or pMHC complexes undergo apoptosis, a
  process known as negative selection
• Thymocytes that survive both positive and
  negative selection migrate from the thymus to
  populate lymphoid tissues and organs as T cells

44
Figure 5-3 part 2 of 2
45
Each thymocyte maturation stage occurs at a
distinct location of the thymus
Young adult5x107 thymocytes produced/day 1.5x10
6 mature cells leave/day
46
Figure 5-3 part 1 of 2
DN (CD4-CD8-) and DP (CD4CD8) Immature
thymocytes are here
Differentiation
More mature SP (CD4CD8-or CD8CD4-) thymocytes
are here
47
Positive Selection

• Positive selection selects T cells that recognize
  peptides on self MHC
• This is to assure that mature T cells can respond
  to antigen-presented on self MHC.
• Self MHC I and II harboring self peptides on
  thymic epithelial cells recognize and activate
  TCRs on some DP thymocytes.
• DP thymocytes should receive this signal within
  3-4 days to survive, otherwise they undergo
  apoptosis.

48
Negative Selection

• Negative selection eliminates T cells with TCRs
  that bind too strongly to self antigen/MHC
  complex (autoreactive cells are removed by this
  process)
• Dendritic cells and macrophages in
  cortico-medullary junction mediate it.
• Negative selection cannot eliminate T cells whose
  receptors are specific for self peptides that are
  present outside of the thymus
• These cells enter circulation, but soon to be
  rendered anergic or unresponsive by other
  mechanims.

49
(No Transcript)
50
Does receptor occupancy explain positive and
negative selection ?
High occupancy Negative selection
Low occupancy Survival
51
Does the TIME of receptor occupancy
explain positive and negative selection ?
52
Stage of maturation can be distinguished by the
expression of specific surface molecules
DN
DP
SP
53
Types of T cells

• Conventional
• Uses ab TCR
• Helper (CD4) and cytotoxic (CD8) T cells
• More abundant and highly specific
• Restricted by classical MHC (I and II) molecules
• Non-conventional
• Uses gd TCR
• Primitive with broad specificity
• Restricted by non-classical molecules

54
CD4 T cells

• T cells with CD4 marker (glycoprotein) represent
  70 of T cells in the periphery
• Named T helper cells
• Play central role in modulating cellular immunity
  via secretion of cytokines that mediate
• B cell activation
• Immunoglobulin secretion (quality)
• Macrophage and dendritic cell activation
• Cellular chemotaxis and inflammation
• Two subsets Th1 and Th2 cells

55
Th1 and Th2 cells

• CD4 T helper cells can be classified into two
  types based on their cytokine profiles T helper
  cell type 1 (Th1) and T helper cell type 2 (Th2).
• Cytokine profile is influenced by several
  factors
• Nature and dose of antigen
• Route of administration
• Type of antigen presenting cell/ costimulation
• Genetic background
• The cytokine profile determines the effector
  function of the helper cell

56
Differentiation of naive CD4 T cells into
different subclasses
57
The nature and amount of ligand determine CD4 T
cell functional phenotype
58
T Helper (CD4 ) Subsets
Antigen
APC
IL-4
Th0
IL-12
FasL
Suicide
Fas
Th1
DR4
Anti-Inflammatory Cytokines
IL-4 IL-10 IL-13 IL-5 IL-6
Pro-Inflammatory Cytokines
IFN-g IL-2 LT
Th1 response Cellular Immunity DTH
Th2 response, Humoral Immunity and Acute
Hypersensitivity
59
Differences between Th1 and Th2 cells
Th1 cell
Th2 cell

• Produces type 1 cytokines
• IL-2, IFN- ?, TNF-a, TNF-ß
• Activates macrophages and DCs for intracellular
  killing of pathogens
• Mediates CMI

• Produces type 2 cytokines
• IL-4, IL-5, IL-10, IL-13
• Provides help to B cells in antibody response
• Mediates allergy and immunity to extracellular
  pathogens, including parasites

60
Cytotoxic T cells

• T cells that express CD8 molecule on their
  surface and they represent 30 of T cells in the
  periphery
• Destroy cells infected by intracellular pathogens
  and cancer cells
• Class I MHC molecules (nucleated body cells)
  expose foreign proteins
• TC cell releases perforin and granzymes, proteins
  that form pores in the target cell membrane
  causing cell lysis and/or apoptosis

61
(No Transcript)
62
Effector molecules of T cell subsets
63
Regulation of the Immune Response

• How does the immune system prevent self
  reactivity while maintaining reactivity to
  invaders/non-self?
• Clonal deletion/inactivation of auto-reactive
  cells
• Regulatory T cells keep potentially pathogenic
  self reactive T cells in check through
  suppressive mechanisms

64
New T cell phenotypes

• Regulatory T cells
• Naturally occurring (CD25 positive)
• Induced (IL-10 and TGF-ß)
• Some NK T cells
• Suppressor T cells
• Th17 cells produce IL-17, role in acute
  inflammation, suppress Th2.

65
Bettelli et al., Nature 2008
66
Th17 and Treg
67
Regulatory T Cells (Treg)

• CD3, TcR, CD4, CD25
• Function
• Suppress the activity of effector Th and Tc cells
• Inhibition is antigen specific and MHC restricted
• Inhibition depends on cellcell contact
• Appear to function in autoimmunity
• Development
• Develop in the bone marrow from DP cells
• Arise as a result of strong binding to self MHC
  and self Ag during negative selection.
  Alternative to cell death or anergy
• Development and maintenance are dependent upon B7

68
What are the requirements for Treg development?

• TCR engagement in the thymus (high affinity but
  not so high as negative selection)
• Higher percentage of thymocytes with high
  affinity TCR in context of auto antigen develop
  into regs
• T cells of appropriate affinity are instructed
  to become Tregs
• Selective sparing of pre-committed cells from
  negative selection or promotion of Treg lineage
  development?
• higher percentage, but not higher absolute number
• Preferential elimination of non-regulatory T
  cells rather than increased production of Tregs
• TCR engagement serves as survival or expansion
  signal of Tregs pre-committed to that lineage.

69
The Treg cell phenotype

• CD4
• Co-receptor for TCR recognition of MHC II/Ag
• CD25 IL-2Ra
• IL-2R component, confers high affinity binding to
  IL-2Rbg
• Key TR growth factor
• CTLA-4 cytotoxic T lymphocyte Ag-4
• Binds to B7s (CD80/86) on APC, acts as
  co-stimulatory molecule for TR (blocking CTLA-4
  inhibits TR)
• GITR glucocorticoid induced TNF related
• protein
• Ligation inhibits TR function (agonist inhibit
  TR, blocking augments TR)
• FoxP3
• Forkhead /winged-helix TF critical for TR
  activity and development
• Unlike surface markers / receptors, TE do not
  express FoxP3

Foxp3
70
Modulation of immune responses by Treg cells

• Treg cells are crucial for the induction and
  maintenance of peripheral tolerance to
  self-antigens
• Treg cells can also suppress immune responses to
• Tumor antigens
• Alloantigen
• Allergens
• Microbial antigens

Sheng Cai