Current Concepts of Immunology

1
Current Concepts of Immunology
NK, NKT and Innate-like T cells (Chapter
2-30-34, 3-19, 5-17-19, 9-23, 11-10 of Janeways
Immunobiology, 7th Edition, Murphy, K., Travers,
P. Walport P.)
Guy Werlen, Ph.D. Associate Professor Dept. Cell
Biology Neuroscience Rutgers, The State
University of New Jersey werlen_at_biology.rutgers.ed
u Office hours Friday 330-500 pm
2
Syllabus
Natural Killer (NK) cells- development-
function in the innate immune system - role as
tumor killers - receptors- ADCC - recognition
of non-classical MHC moleculesInnate-Like T
cells - NK T cells- IEL ?? T cells- IEL CD8??T
cells
3
NK cells development
Bone marrow

• In the bone marrow, a common lymphoid progenitor
  gives rise to the antigen-specific lymphocytes of
  the adaptive immune system (B and T cells), as
  well as to a type of lymphocytes that is antigen
  non-specific the Natural Killer (NK) cell
  lineage (express the NK lineage marker, NK1.1).
• - As NK cells are antigen independent, they
  belong to the innate immune system.
• Mature NK cells that circulat in the blood
  stream are larger than B and T cells.
• They contain a distinctive granular cytoplasm.
  The release of the lytic granules contributes to
  the killing of cells infected with distinctive
  viruses, such as herpes virus.
• NK cells also contribute to the killing of
  abnormal tumor cells, without prior immunization
  or activation.

Blood
4
NK cells contribute to the cytotoxic response
CTL NK cells

• Mature NK cells that circulate in the blood are
  larger than B and T cells.
• They contain a distinctive granular cytoplasm,
  that is composed of lytic vacuoles.
• - Similar than CTL, the lytic granules of NK
  cells contain the proteins perforin, granzyme and
  granulysin.
• The release of the lytic granules contributes to
  the killing of cells infected with distinctive
  viruses, such as herpes virus.
• NK cells also contribute to the killing of
  abnormal tumor cells, without prior immunization
  or activation.

5
IFN released by virus-infected cells activate NK
cells

• Most virally infected cells will secrete
  interferons.
• The interferons, INF-? and INF-? are
  multifunctional proteins produced in response to
  the activation of TLR-3 by long viral
  double-stranded RNA molecules.
• NK cells are rapidly activated by INF-? and
  INF-?? They specifically destroy infected but not
  normal cells.
• INF-? and INF-??also induce increased expression
  of MHC Class I on all cells including the
  infected cells. Viral antigen presentation on
  infected cells activates T-cell mediated killing
  and clearing of the viral infection.

6
Release of cytotoxic granules at the site of
contact with infected cells

• First contact between a CTL or NK cell with
  infected cells is via non-specific binding of
  adhesion molecules (LFA-1 (blue) on T and NK
  cells with ICAM-1 or ICAM-2 (brown) on target
  cells). This makes a channel between the target
  and the cytotoxic cell.
• Specific antigen/MHC class I recognition by TCR
  on CTL, or engagement of the NKs natural
  cytotoxic receptors (NCR) (green) by non-MHC
  ligands (orange) on the surface of the target
  cell. This results in a polarization of the cell
  the actin cytoskeleton (green staining in the
  immunofluorescence microscopy) at the site of
  contact is reorganized as to aligning the
  microtubule-organizing center (MTOC), as well as
  the secretory apparatus, including the Golgi
  (GA). The GA-derived lytic granules (stained in
  red in the photomicrograph) are specifically
  directed onto the target cell.
• The content of the granules is directly released
  onto the target cell.

7
NK cells express sensing receptors for self-MHC
class I
CTLs recognize antigens presented on MHC class I
molecules and kill infected cells.
NK cells function is blocked by the binding of
MHC class I molecules on normal cells.
Killer-cell immunoglobulin-like receptors (KIRs)
or the lectin-like receptor NKG2 expressed on the
surface of NK cells recognize MHC class I
molecules. This association triggers an
inhibitory signaling cascades inside the NK cell
that overrules any potential activation signal.
8
Target killing by NK cells
This inhibitory signal is lost when target cells
do not, or only weakly express MHC class I
molecules. Target killing ensues due to the
activation of killer receptors (green) that are
expressed on NK cells. Many virus negatively
affect the expression of MHC class I molecules.
The mechanisms of infected cell recognition and
killing by NK cells is thus more efficient than
antigenic recognition by CTL.
9
Why are NK cells prominent tumor killers?
Similar to many pathogens, tumor cells can escape
the adaptive immune system, by downregulating the
expression of MHC class I. This makes them more
susceptible to NK cells.

• - The regression of transplanted tumors in a
  normal mouse model (blue line) is largely due to
  the action of CTLs recognizing tumor antigens
  presented on MHC class I (right panel). Albeit
  the presence of NK cells, this regression is
  absent in nude mice (red line) in which CTLs do
  not develop.
• Tumor variants that express low levels of MHC
  class I become susceptible to NK cells,
  especially in nude mice (have higher levels of NK
  cells than wild type mice). Thus tumors that are
  sensitive to NK killing grow less well in nude
  than normal mice (central panel).
• - Transfection of MHC class I genes resulting in
  high expression of this protein restores NK cell
  resistance but susceptibility to CTL in normal
  mice (left panel blue line).

10
Genomic clusters of genes regulating the activity
of NK cells

• The leukocyte receptor complex (LRC) is a large
  cluster of genes that encode a family of Ig
  domain-like proteins, such as the KIR (killer
  cell immunoglobulin-like receptors), or the
  natural cytotoxic receptor (NCR), NKp46.
• The NK receptor complex (NKC) is a second
  cluster that mainly encodes a family of
  lectin-like receptors (KLR).
• - Both clusters are present in humans and mice,
  but mice lack the KIR genes.
• ??Some NK receptor genes, such as the NCRs, NKp30
  NKp44 are found outside of these 2 major
  clusters

11
NK receptors Defense is the best offense

• While both KIRs and KLRs sense the presence
  (absence) of MHC class I molecules, activating as
  well as inhibitory receptors are found in both
  families of receptors.
• The KIRs are subdivided according to the number
  of immunoglobulin-like domains (2 or 3 domains)
  and the length of their cytoplasmic tail Short
  tail activating receptors
• Long tail inhibitory receptors
• The KLR are heterodimers of CD94 associated with
  a NKG2 molecule. Six distinct NKG2 isoforms exist
  in humans.
• NKG2C/CD94 activating receptor
• NKG2A/CD94 inhibitory receptor
• NKG2B/CD94 inhibitory receptor
• NKG2D homodimer activating receptor.

12
iTIM and iTAM are the Ying and Yang of immune
cells

• Immunoreceptor tyrosine-based inhibitory motifs
  (ITIMs consensus sequence S/I/V/LxYxxI/V/L) and
  immunoreceptor tyrosine-based activation motifs
  (ITAMs YxxI/Lx6-12YxxI/L) are motifs found in a
  large number of receptors or adaptor proteins.
  Phosphorylated tyrosines of ITAMs serve as
  docking sites for tandem SH2 domains of Syk
  family kinases, whereas phosphorylated tyrosines
  of ITIMs recruit tyrosine phosphatases, such as
  SHIP and SHP. Signaling through ITAM-bearing
  receptors usually results in cell activation,
  while engagement of ITIM-bearing receptors is
  usually inhibitory.
• The extension of the KIRs cytosolic tail
  (KIR2DL and KIR3DL) contains ITIMs, which
  contribute to the inhibitory signal upon MHC
  class I ligation.
• KIRs with short cytoplasmic domains as well as
  the activating receptors of the KLR family
  associate with the adaptor protein DAP-12, CD3?
  or the Fc receptor ? chain that bear ITAMs and
  similar as the TCR/CD3 bind the tyrosine kinase
  ZAP-70 and Syk following phosphorylation..

13
Fc receptors a cell- and ligand-specific family
Fc receptors exist in different flavors on the
surface of distinct immuno cells. As their names
are suggesting they all bind Fc regions of
specific immunoglobulin classes and contribute to
the elimination of pathogens. NK cells
specifically express Fc?RIII.
14
AD CC or Hells Bells for NK target cells
IgG antibody-coated target cells are rapidly
killed by NK cells expressing the FC?RIII
receptors. As it requires a fully functional
adaptive immune system, this mechanism known as
antibody-dependent cytotoxicity (ADCC) is
probably a relatively recent addition to the
repertoire of NK cell killing mechanisms. Its
role in the defense against bacteria or viruses
has not yet been fully established..
15
.. ADCC as a therapeutic means to kill tumor
cells
Among other powerful immuno-cancer therapies,
tumor-specific antibodies of the correct isotypes
can by used to eliminate malignant cells via ADCC
(left panel).
16

• Besides KIRs, KLRs and FC?RIII, NK cells express
  receptors that more directly bind ligands derived
  from pathogens, the NCRs (natural cytotoxicity
  receptors). The NKp molecules are Ig-like
  receptors, while NKG2D belongs to the NKG2 family
  of C-type lectin receptors.
• NKp46 recognizes heparan sulfate proteoglycans
  as well as some viral proteins.
• In contrast to the other members of the NKG2
  family that heterodimerize with CD94 and mainly
  recognize the (human) MHC class I molecule, HLA-E
  (Qa-1, mouse), NKG2D homodimerizes and binds
  non-self, MHC-like molecules.
• Human NKG2D binds the intracellular adaptor,
  DAP-10 leading to the activation of the
  phosphatidylinositol-3-kinase (PI-3K) pathway,
  without ITAM phosphorylation, as DAP-10 is
  deprived of these motifs. In mice, 2 alternative
  splice variants of NKG2D exist that bind
  respectively DAP-10 or the ITAM containing
  DAP-12, leading to the activation of distinct
  signaling pathway.

17

• Unlike the other NKG2 family members that
  recognize MHC class I molecules, NKG2D receptors
  are activated by the non-classical MHC class 1b
  molecules, MIC-A, MIC-B or RAET1.
• The expression of these proteins is induced by
  stress, such as infection, metabolic or heat
  stress. Thus recognition of those proteins by
  NKGD2 is a generalized danger signal to the
  immune system.
• The structure of their NKG2D binding domains,
  resemble to the ?1 and ?2 domains of MHC class I
  molecules. They do not associate with
  ?2-microglobulin (?2-m).
• They have low to moderate polymorphism, but do
  not bind a peptide ligand (direct binding to
  NKG2D).
• Mice do not express equivalents of MIC proteins
  and NKG2D seem only to be activated by ligands
  similar to the human RAET1 family (known as
  retinoic acid early inducible 1 (Rae1) protein
  family).
• Some of the RAET1 family members are ligands for
  the cytomegalovirus protein, UL16 and are
  therefore known as UL16-binding proteins (ULBPs).

18
NONclassical MHC class I molecules

• Many proteins with homologies to MHC Class I
  molecules are encoded within region I of the MHC
  locus.
• They are pooled in the Class 1b subgroup. Many
  of these proteins bind to ?2-m and have a role in
  innate immunity by regulating the activation of
  NK cells or innate-like T cells.
• HLA-E (mouse Qa-1) bind MHC leader peptides
  (determinant modifiers) and upon engagement of
  NKG2A/CD94 or NKG2C/CD94 heterodimers blocks NK
  cell activation.
• Similarly, HLA-G expression on fetus-derived
  placental cells prevents NK cell activation and
  destruction of the placental cells that do not
  express classical MHC class I molecules.
• However, not all have a function in the immune
  system, such as the protein encoded by the mouse
  gene, M10 which is recognized by pheromone
  receptors in the vomeronasal organ (influence
  mating preferences).
• Others, have specific functions due to their
  specific expression pattern or low polymorphism
  (the mouse H2-M3 molecule presents N-formylated
  peptides to CTL).

19

• Many Class 1b molecules are encoded on other
  chromosomes than the MHC locus, such as the NKG2D
  ligands, ULBPs.
• The protein product of HFE (ex HLA-H) is
  expressed on cells of the intestinal tract and
  has a function in iron metabolism. Its mutation
  causes iron-storage diseases.
• The CD1 family of MHC class I-like molecules
  presents microbial lipids to CD1-restricted T
  cells. CD1 molecules are expressed on DC,
  monocytes and some thymocytes. While, they are
  similar as MHC class I molecules in structural
  organization and association to ?2-m, CD1 family
  members behave like MHC class II molecules (not
  retained within the ER by association with TAP,
  but targeted to vesicles, where ligand binding
  takes place). In addition, CD1 molecules have a
  hydrophobic channel that specifically binds
  hydrocarbon alkyl chains of glycolipids.
• . Group 1 comprising CD1a, CD1b and CD1c bind
  microbial derived glycolipids, phospholipids as
  well as lipopeptides.
• . Group 2 only contains CD1d that mainly binds
  self lipid antigens, such as sphingolipids and
  diacylglycerols.
• . CD1e is intermediary of the 2 groups and binds
  either glycolipid molecules.
• - CD1-restricted T cells do not express CD8 or
  CD4 (can not recognize classical MHC class I or
  class II molecules).
• Divers repertoire of ??TCR bind lipid ligands
  presented by group 1 CD1 molecules.

20
Innate-like T cells
Innate-like lymphocytes (ILL) are at the X-road
of innate and adaptive immunity they express
receptors commonly found on the surface of NK
cells, such as members of the NKG2 family and the
NK lineage receptor NK1.1, as well as rearranged
BCR or TCR (but with very little diversity).
ILL do not undergo clonal expansion before
responding effectively to antigen.
21
NK T cell are mainly generated in the thymus
DN
NK T
DP
NK T cells are generated in the thymus from CD4
CD8 DN precursor thymocytes. While most of the
thymocytes of this lineage never express CD4 or
CD8, their differentiation is CD1d/lipid ligand
restricted.
SP
22
NK T cell a jack of all trades
While NK T cells primary function is
cytotoxicity (trait of NK and CTL lineages) they
also have helper characteristics as the
secretion of IFN-? or IL-4 induces T helper cells
to differentiate into either TH1 or TH2 cells.
23

• - Unlike the major subset of T cells that express
  high diversity ?? or ?? TCR (cells commonly found
  in lymphoid tissues), intraepithelial (IEL) ?? T
  cells express TCR with very limited diversity.
• - They reside mainly in the epithelium of the
  female reproductive tract and the skin.
• Based on their limited diversity and the lack of
  recirculation, one thinks that IEL ?? T cells
  directly recognize ligands, such as heat shock
  proteins, phospholipids, MHC class Ib molecules
  and unorthodox nucleotides that are derived from
  the epithelium in which they reside but that are
  expressed by the epithelial cells only upon
  infection.
• This would distinguish IEL ?? T cells from the
  rest of the high diversity T cells that only
  recognize pathogen derived ligands bound on self
  MHC class I and class II molecules.

24
IEL ??? T cells mature in the thymus
DN cells include precursors that will generate a
minority of T cells that express the ??TCR (never
express CD4 or CD8 co-receptors even as mature T
cells in the periphery) and a majority of cells
that will express a ??TCR together with either
CD4 or CD8. ?? T cells do not undergo the
self-MHC restricted selection process that
specifies ?? T cells.
DN
DP
SP
25
Signals trough the ??TCR and the pre-TCR compete
to determine thymocyte lineage
The ?, ? and ? TCR loci are simultaneously
rearranged in DN thymocytes. If a complete and
functional ?? TCR is formed before successful ?
gene rearrangement has led to the production of a
pre-TCR, the thymocyte commits to the ?? lineage
by shutting of further rearrangement of ?-chain
genes (negative signal in the cartoon). In this
case, ?? T cell develop and migrate to the
periphery (left panels). In contrary, if a
functional ?-chain is rearranged before the ??
loci, signals trough the pre-TCR commit the
thymocyte to the ?? lineage by shutting off
further rearrangement of the ?? and ?? chain
genes (negative signal in the cartoon). In this
case, ?? T cell develop and migrate to the
periphery (right panels).
26
IEL??? T cells are generated in waves during
fetal development
IEL ?? T cells bearing distinct rearranged V?
segments (V?5 and V?6) develop in waves before
birth of a mouse fetus. V?5-bearing cells become
selectively established in the epidermis, while
V?6-bearing cells home the epithelium of the
reproductive tract. At each site the ?? T cell
population is homogenous since their distinct ?
chains pair with the same ? chain. After birth,
T cell of the ?? lineage become dominant, while
the few ?? produced are now more heterogeneous
expressing TCRs with a great deal of junctional
diversity.
27
Lymphocytes of the intestinal mucosa

• ??The lamina propria (center panel) is colonized
  by conventional effector or memory CD4 and CD8??
  T cells (CD4CD8 ratio 31) that express the
  integrin ?4?7 (selectively binds MAdCAM-1
  expressed on the endothelium of blood vessels in
  mucosal tissues),
• - 80 of the lymphocytes in the epithelium (right
  panel) express CD8 of which 50 are unusual T
  cells as the express CD8?? homodimers (ILL CD8??
  T cells).
• The majority of intestine IEL express ???TCR,
  while a minority is from the ?? lineage. They
  express the integrin ?E?7 that selectively binds
  to E-cadherin on epithelial cells
• All T cells from the intestine mucosa express the
  chemokine receptor CCR9, which attracts them into
  the tissue from the bloodstream.

28
CD8?? IEL T cells are also generated in the thymus
DN
Development of ab thymocytes from DN cells occurs
via a CD4CD8 double positive (DP) stage in
which both coreceptors are expressed. DP
thymocytes undergo a selection process in which
the self-reactive cells die (negative selection)
and only a minority of cells that recognize a
self-ligand will survive (positive selection) and
differentiate into either the T Helper linage
that downregulates CD8 or CTL lineage that
downregulates CD4. While most T cells of the CTL
lineage express a CD8???coreceptor, some express
a CD8?? homodimer that will not bind a
self-ligand in the periphery.
DP
SP
CD8??
29
Modulation of signals leading to the generation
of CD8?? thymocytes
Low affinity ligand
High affinity ligand
??TCR CD8??

• High affinity ligand-stimulation induces a fast
  en transient ERK activation that peaks before
  maximal JNK and p38 activation. This leads to
  thymocyte negative selection and death or to the
  generation of regulatory T cells with
  autoreactive potential, such as the CD8?? IEL.
• In contrary low affinity ligands induce a slow
  and sustained ERK activation that leads to
  positive selection and thymocyte differentiation.
  Modified from Werlen et al, 2003, Science 299,
  1859-1863.
• - The ?-galactose binding protein, Galectin-1
  (gal-1) expressed on thymic cortical cells
  modulates ligand binding to the TCR and enforces
  negative selection, while opposing positive
  selection. Its absence leads to increased
  differentiation of CD8aa IEL. (Liu et al. 2008,
  Blood, 112120-130).

30
Role of conventional and ILL CD8 T cells in the
gut epithelium a killer tandem
Conventional IEL CTLs kill virally infected
epithelial cells that present viral peptides on
their surface. Dual mode killing via release of
perforin granzyme, as well as Fas-dependent
death pathway.
Epithelial cells that are stressed upregulate the
expression of MIC-A and MIC-B. These
non-classical MHC class Ib molecules bind NKG2D
of IEL. The non-classical MHC class Ib molecule
TL binds directly CD8???expressed on ILL CD8 T
cells. The ILL cells will not bind ligands
composed of viral peptides since the expression
of CD8?? prevents binding of self-MHC class I
molecules.
31
Role of CD8 ILL in Celiac disease
Celiac disease is a antigen-specific
immunopathology of the upper small intestine
directed at gluten, a complex of proteins present
in wheat, oats and barley. The disease has a
strong genetic component since 95 of patients
express the HLA-DQ2 class II MHC. Peptides
normally produced from gluten do not bind MHC
class II molecules. Peptides modified by the
enzyme, tissue transglutaminase (tTG) are
specifically recognized by HLA-DQ2, triggering
the activation of antigen-specific CD4 T cells
and the destruction of epithelial cells.
Gluten peptides can activate eptithelial cells of
the intestine to upregulate the expression of the
nonconventional MHC class Ib molecules MIC or TL.
Ensues activation of IEL CD8 T cells and the
destruction of the epithelium independently from,
or in conjunction to the antigen-specific CD4 T
cell response.