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Strategie evolutive
dei meccanismi di
difesa contro le
infezioni
Andrea
Cossarizza
Dept. Biomedical Sciences
Univ. of Modena
& Reggio Emilia
MAIN TOPICS
NKT cells
- distribution and characteristics
- biological role
MAIN TOPICS
NKT cells
- distribution and characteristics
- biological role
ANTIGEN PRESENTATION by CD1
- molecules and distribution
- functional role
MAIN TOPICS
NKT cells
- distribution and characteristics
- biological role
ANTIGEN PRESENTATION by CD1
- molecules and distribution
- functional role
TOLL LIKE RECEPTORS
- ligands and role
- evolutionary aspects
- counteractions by pathogens
MAIN TOPICS
NKT cells
- distribution and characteristics
- biological role
ANTIGEN PRESENTATION by CD1
- molecules and distribution
- functional role
TOLL LIKE RECEPTORS
- ligands and role
- evolutionary aspects
- counteractions by pathogens
INNATE IMMUNE SYSTEM
Because of its critical role in
initiating and orchestrating a
productive immune response, it is
imperative to obtain a detailed
understanding of the microbial
determinants that are recognized by
different components and cells of
the innate immune system.
One member of the innate
immune system, the natural
killer T (NKT) cell, is activated
during bacterial infections.
NATURAL KILLER T CELL (NKT)
 NKT cells constitute a unique subpopulation of T
lymphocytes that is highly conserved, and well evident in
both human and murine species.
 The term 'NKT cells' was first used to describe a small
subset of the T lymphocytes that coexpressed some
markers traditionally associated with NK cells.
 The most prominent of these markers was the NK1.1
antigen (NRK-P1/CD161 in humans), which has regularly
been used in conjunction with TCR expression to
phenotypically identify NKT cells.
 At first glance, NKT cells may be easily mistaken as
being part of the adaptive immune system.
T CELL RECEPTOR EXPRESSION
Like conventional T cells, NKT cells express T-cell receptors
that are generated via somatic DNA rearrangement.
T CELL RECEPTOR EXPRESSION
The TCRs of most NKT cells are semiinvariant, consisting of Vα14–
Jα18/Vβ8.2 chains in mouse and
homologous
Vα24 – Jα18 / Vβ11 chains in
human (iNKT cells).
NATURAL KILLER T CELL
 In humans, only 0.2% of peripheral blood T
cells are NKT cells
 They are also present in the human liver and
play a crucial role in the control of cells infected
by hepatitis virus
 NKT cells express markers associated with
recently activated or memory T cells
CO-RECEPTOR EXPRESSION
CD4
 Human and mouse NKT cells segregate into CD4+CD8− and
CD4−CD8− (double negative, DN) cell subsets, which differ in
their functional properties.
 In humans, about 40 to 60% of invariant NKT cells are CD4+,
with high donor-to-donor variability, whereas the remaining cells
lack CD4 expression.
 In humans, CD8α expression is common, but only very few
CD8β+ NKT cells exist (CD8 expression is even absent in mice)
CD8
 The CD4+ subset potently produces both Th1 and Th2
cytokines
 DN population selectively produces the Th1 cytokines (IFN-)
and TNF-a and preferentially upregulates perforin in response to
IL-2 or IL-12
ANTIGENS RECOGNITION: CD1d
 NKT cells are activated by glycolipid antigens presented in
the monomorphic MHC I-like molecule CD1d highly conserved
among mammalian species.
CD1 MOLECULES
CD1 molecules comprise a family of
glycoproteins that resemble class I MHC
molecules in their domain organization.
Humans possess one
of each of the five
CD1 isoforms, located
on chromosome 1, in
the order shown.
Mice and rats appear to have duplicated
CD1d and lost the genes for CD1a, CD1b,
CD1c and CD1e, possibly in a
chromosomal translocation event.
Ag presentation by CD1
The discovery of molecules capable of presenting lipid
antigens and of the T cells that recognize them has
opened a new dimension in our understanding of cellmediated immunity against infection.
Like MHC Class I molecules, CD1 isoforms (CD1a, b,
c and d) are assembled in the ER and sent to the cell
surface.
However, in contrast to MHC molecules, CD1
complexes are then re-internalized into specific
endocytic compartments where they can bind lipid
antigens.
Assembly of nascent CD1 molecules in ER is
assisted by chaperones (orange). Microsomal
triglyceride transfer protein (MTP, blue)
assists CD1 loading with ER-resident selfGSL (red).
In LE/Ly, LTP (light blue) extract lipids from
membranes and participate in loading and
unloading of CD1 molecules. Soluble CD1e is
involved in processing of large microbial
glycolipids (in green) by binding and offering
them to hydrolases (yellow).
Upon internalization of bacteria (green cells),
microbial lipids are released into phagosomes
and loaded onto CD1 molecules.
Infection, by altering self-lipid metabolism
facilitates CD1–self-lipid complex generation
and activation of autoreactive T cells.
De Libero et al. Eur J Imm, Oct. 09
Assembly of nascent CD1 molecules in ER is
assisted by chaperones (orange). Microsomal
triglyceride transfer protein (MTP, blue)
assists CD1 loading with ER-resident selfGSL (red).
In LE/Ly, LTP (light blue) extract lipids from
membranes and participate in loading and
unloading of CD1 molecules. Soluble CD1e is
involved in processing of large microbial
glycolipids (in green) by binding and offering
them to hydrolases (yellow).
Upon internalization of bacteria (green cells),
microbial lipids are released into phagosomes
and loaded onto CD1 molecules.
Infection, by altering self-lipid metabolism
facilitates CD1–self-lipid complex generation
and activation of autoreactive T cells.
De Libero et al. Eur J Imm, Oct. 09
Assembly of nascent CD1 molecules in ER is
assisted by chaperones (orange). Microsomal
triglyceride transfer protein (MTP, blue)
assists CD1 loading with ER-resident selfGSL (red).
In LE/Ly, LTP (light blue) extract lipids from
membranes and participate in loading and
unloading of CD1 molecules. Soluble CD1e is
involved in processing of large microbial
glycolipids (in green) by binding and offering
them to hydrolases (yellow).
Upon internalization of bacteria (green cells),
microbial lipids are released into phagosomes
and loaded onto CD1 molecules.
Infection, by altering self-lipid metabolism,
facilitates the generation of CD1–self-lipid
complex and the consequent activation of
autoreactive T cells.
De Libero et al. Eur J Imm, Oct. 09
Ag presentation by CD1
CD1 molecules are differentially expressed by a variety of cell
types including DC, B cells, monocytes, Langerhans cells,
stellate hepatic cells, epithelial cells, microglial cells and
keratinocytes.
CD1a, CD1b, CD1c and CD1d molecules reach the plasma
membrane and are involved in lipid presentation to T cells,
whereas CD1e remains intracellular and is involved in lipid
processing.
Lipid-specific T cells express a variety of TCR heterodimers, with
the exception of invariant NKT (iNKT) cells. Lipid-loaded CD1
molecules delivered to the cell surface are surveyed by CD1restricted T cells expressing ab or gd TCR.
CD1 PRESENT A WIDE VARIETY OF LIPIDS
Endogenous cellular lipids, foreign lipids derived from intracellular
parasites and extracellular lipids of self or foreign origin.
The degree to which different CD1 isoforms are specialized for binding
structurally distinct lipids remains unclear
CD1a
present a mycobacterial lipopeptide in addition to glycolipids
CD1b
accommodates lipids with very long alkyl chains (e.g. C80;
mycolates that are components of mycobacterial cell walls)
CD1c
present mycobacterial isoprenoid lipids that contain unusual
alkyl chains consisting of repeating branched, unsaturated
units.
Some lipids, including phospholipids and sphingolipids, have been shown
to bind multiple CD1 isoforms including CD1a, CD1b, CD1c and CD1d
Structures of self-lipid antigens.
iGb3: isoglobotrihexosylceramide; PG: phosphatidylglycerol;
PE: phosphatidylethanolamine; PI: phosphatidylinositol.
Structures of microbial lipid antigens.
a-galactosylceramide (a-GalCer) extracted from A. mauritianus.
BbGL-II: Borrelia burgdorferi diacylglycerol;
GMM: glucose monomycolate; GroMM: glycerol monomycolate.
iNKT cell activation
During infection, iNKT cells are rapidly elicited. Activated
iNKT cells can produce a vast array of cytokines (IFN-,
IL-4, IL-2, IL-5, IL-10, IL-13, GM-CSF, TNF- …...) that
affect the innate and the adaptive immune response
Ag presentation by CD1
hot points:
The capacity of the immune system to recognize lipid
antigens relies on a series of biochemical and biological
characteristics of lipid molecules.
These are associated with the structure of lipids, which
affects their bioavailability, type of trafficking and
capacity to associate with CD1 molecules.
It also depends on the structure of CD1 molecules that
have evolved different antigen-binding pockets and
trafficking capacity.
Ag presentation by CD1
hot points:
An important difference between MHC and CD1 antigen
presenting molecules is that CD1 are functionally nonpolymorphic and this results from selective pressure
dictated by the structure of microbial lipid antigens.
While peptides lose their MHC-binding capacity by
changing single amino acids, lipids are much more
constrained in changing their CD1-binding moieties
because these modifications are often not compatible
with their biological function.
MAIN TOPICS
NKT cells
- distribution and characteristics
- biological role
ANTIGEN PRESENTATION by CD1
- molecules and distribution
- functional role
TOLL LIKE RECEPTORS
- ligands and role
- evolutionary aspects
- counteractions by pathogens
Toll-like receptors (TLRs)
A family of transmembrane receptors that
have been highly preserved throughout
evolution.
Neighbour-joining molecular tree of the
extracellular domains of mammalian TLRs
Sequence evolution in the extracellular domain after
gene duplication might be the key to understanding how
the ligand binding affinity evolves.
Positive pressure
Purifying selection
w = dN/ds
(non-synonymous/
synonymous
substitution ratio)
Analysis of a dataset of 22 mammalian TLR2 sequences (black line),
compared to sequences of ten ruminant TLR2 sequences (red line)
Toll-like receptors (TLRs)
A family of transmembrane receptors that
have been highly preserved throughout
evolution.
They selectively recognize a broad
spectrum of microbial components
(PAMPs: Pathogen Associated
Molecular Pattern) and endogenous
molecules released by injured tissue
Different TLR recognize different PAMPs
Different TLR use different intracellular pathways
WHAT HAPPENS AFTER TLR TRIGGERING
ANTI-IMMUNE EVOLUTION
OF PATHOGENS
Anti-interferon
mechanisms
Activation of the interferon response triggered
by the detection of viral PAMPs
Activation of the interferon response triggered
by the detection of viral PAMPs
Activation of the interferon response triggered
by the detection of viral PAMPs
Viral evasion of TLR signalling
Viral evasion
of retinoicacid-induciblegene-(RIG) ilike receptor
signalling
Inhibition of interferon-regulatory factor 3
(iRF3) and iRF7 by viral proteins
CONCLUSIONS
• DURING EVOLUTION, THE IMMUNE
SYSTEM HAS DEVELOPED SEVERAL
STRATEGIES TO FIGHT INFECTIONS;
• INFECTIVE AGENTS HAVE DEVELOPED
SEVERAL STRATEGIES TO COPE WITH
THE IMMUNE SYSTEM.
• THE GENERAL SURGEON WAS NOT
RIGHT…… THERE WILL BE ALWAYS
SOMETHING TO DO – INCLUDING
BOOKS TO WRITE - FOR US!
Thanks
for your
attention