We are in the lymph node, where
our B cells, Dendritic Cells, and T
cells are waiting.
Remember, naïve B cells were
recruited to the cortex, and form
primary follicles with FDC.
Naïve T (TH and TC) cells were
recruited to the paracortex, where
they associate with DC and FRC
Any antigens/pathogens in the
extracellular fluid were also
carried with the flow of lymph to
the node.
Dendritic cells and macrophages secrete a chemokine called CCL18 to
attract T cell to them. The T cell will use the TCR to scan MHC-Ag on the
DC, to see if the DC is presenting an antigen fragment that has epitopes
that the T cell can bind to. If there is binding between the TCR and MHCAg, cell adhesion molecules (the ICAMS in the picture below) will help
hold the two cells together.
T cell receptor (TCR) complex
•
•
•
•
•
The TCR complex contains the TCR, which binds the Antigen MHC, and
additional peptides, which form a protein called CD3.
CD3 is required because the cytoplasmic region of the TCR peptides are
not long enough for signal transduction. CD3 fills the signal transduction
role
CD3 is really three heterodimer pairs of peptides.
CD3 has long enough cytoplasmic tails for signal transduction
The tails contain ITAM motif-there are amino acid sequences that recruit
signaling kinases.
The TCR/CD3 complex also is associated with either CD4 (TH) or
CD8 (TC). These are co-receptors, that bind to MHC.
The cytoplasmic tail of CD4/8 associates with the signaling
intracellular domain of CD3.
We will focus on activation of naïve TH cells for now.
Interaction between the TH cells and the APC will send
three signals to activate the naïve TH.
The first signal, called Signal 1, is when the TCR/CD3
complex recognizes and binds MHC II-Ag. CD4 binds
to MHC II. The signaling cascade is shown in a later
slide. This signaling cascade starts the activation
process.
Signal 2 is caused by CD28 on the T cell binding to
co-stimulator B7 on the APC. B7 is a dimer of B7.1
and B7.2. (B7.1 is also known as CD80. B7.2 is also
known as CD86). This activates more intracellular
signaling, and will lead to synthesis in the T cell of IL-2
and the IL2 receptor (among other things). IL-2
stimulates proliferation and survival.
Signal 3 is caused by the cytokines secreted by the
APC that tell the TH what type of effector cell it should
become.
Next we will go into each signal in more detail
• Signal 1:
• The ends of the TCR bind the Ag-MHC
with high specificity. We will discuss the
binding sites later in the course. If it is a
TH cell, it binds MHC II. If it is a TC cell, it
binds MHC I.
• If there is binding between the TCR and
MHC-Ag, cell adhesion molecules will
help hold the two cells together.
• The CD4 (for TH), or CD8 (for TC) coreceptor binds to MHC II (CD4) or I
(CD8). This binding is outside the Ag
binding areas.
• When CD4/8 binds, it brings a kinase,
called p56Lck close to the cytoplasmic
tails of the CD3 peptides. p56Lck then
phosphorylates the CD3 tails to start
Signal 1.
•
•
•
•
Signal 1, continued:
Phosphorylation of CD3 tails by p56Lck
creates a docking site for another protein
kinase called ZAP-70.
Once bound to the phosphorylated CD3
tails, ZAP-70 phosphorylates adaptor
molecules that recruit components of
several additional signaling paths.
Paths include PLCg, Ca++, PKC, MAP kinase.
.
Pathway can
activate
transcription
factors
•
The Ca++ release caused by Signal 1
stimulates transport of a transcription
factor, called NFAT, into the nucleus,
where it binds to specific promoters.
•
The genes that are activated by NFAT
depend of whether or not Signal 2
happens. Signal 2 should happen at the
same time as Signal 1.
•
Without Signal 2, NFAT transcribes
genes, such as GRAIL (Gene Related to
Anergy In Leukocytes) that will lead to a
state called anergy.
•
What is ANERGY? Anergy is a state of
non-responsiveness-the T cells can’t
respond to activation signals, and so they
just sit there and don’t do anything. They
eventually die.
•
GRAIL disrupts the adhesion between T
cells and the APC. (it marks adhesion
proteins for degradation-its an E3
ubiquitin ligase, for those of you who like
to remember Cell Biology)
•
Without Signal 2, TH cells are
unresponsive.
MHC-Ag
Signal 1
Nature Reviews Immunology 14, 435–446 (2014)
•
Signal 2, the co-stimulatory signal, is
caused by CD28 on T cells binding to B7
on APC.
•
B7 binding to CD28 activates additional
kinases-including the MAP kinase
pathway. This results in activation of
additional transcription factors.
•
Instead NFAT causing cells to making
GRAIL, and become anergic, when Signal
1 combines with Signal 2, the two paths
converge to activate additional
transcription factors, including NF-kB
and AP-1, that work with NFAT to turn on
the genes for IL-2 and the IL-2 receptor
(IL-2R, also known as CD25).
•
If TCR-CD3-CD4/CD8 paths are activated
without the CD28-B7 interaction, the T
cells enter a state of ANERGY, which
means they can’t proliferate, and are
generally non-responsive.
•
Generation of anergic TH cells should
help prevent autoimmunity. B7 is only on
antigen presenting cells, and it is
increased by signaling from PRRs. If
there are no PAMPS, then. There is little
B7. No T cell activation.
Signal 2
B7
MHC-Ag
Signal 1
Nature Reviews Immunology 14, 435–446 (2014)
This picture shows some of the adhesion molecules that strengthen the
connection between the T cell and APC. This is what GRAIL gets rid of.
When both Signal 1 and Signal 2 take
place, IL-2 is secreted by the T cell, and
then the IL-2 binds to the Il-2R that was
also made by the T cell. Consider this to be
Signal 2a.
TCR-MHC II/Ag, CD4-MHC II activate
Signal 1, and CD28-B7 activates Signal 2.
IL-2 binds to the IL-2 receptor (CD25).
This starts a another signaling path that
causes the T cells to enter the cell cycle
and to proliferate and begin differentiation
into effector cells or memory cells.
In addition to inducing the T cells to
proliferate, IL-2 is also a survival signal,
and at this stage, if there is no IL-2, T cells
die by apoptosis.
What happens as result of Signals 1 and 2
Signal through a JAK-STAT pathway, interfaces with MAP Kinase (ERK)
pathway, which starts the cell cycle, and the PDK-1 path, which affects
apoptosis regulators to allow survival.
Signal three is the differentiation signal. It
is started when cytokines secreted by the
APC or other nearby cells bind to specific
cytokine receptors on to the T cell.
In many cases, the APC secretes specific
cytokines that work using the JAK-STAT
signaling pathway.
The STATs, which are specific for each
cytokine receptor, turn on genes encoding
key regulatory transcription factors (see
next slide), which then turn on the genes
specific to a type of TH effector cells, or
memory cell.
In many cases, the PRR on the APC that
were activated by the antigen/PAMPS
determine which cytokines are secreted for
Signal 3.
Signal 3
Cytokine for signal 3 from
APC
Key regulatory
Transcription factor
Genes that are turned
on-the genes encode
these cytokines that are
secreted by the effector
TH cells.
The top row on the figure shows the cytokines that act as Signal 3. Note that some of the Signal 3
cytokines are inflammatory cytokines, which were made by the APC in response to TLR signaling.
Naïve TH cells differentiate into a type of effector TH cells (e.g. TREG, TFH,TH17, TH1, and TH2). We will
discuss these in later slides and also later in the course.
Each effector TH cells is characterized by a distinct regulatory transcription factor, that helps turn on
the genes needed for the cell to do its job. Among these gene are cytokines that are secreted by
the TH effector cells-each type has its own distinct set of cytokines that it secretes. We will discuss
these effector functions in detail a bit later in the course. Right now we need to talk more about
Signal 3.
O’Neill LA, Bryant CE, Doyle SLTherapeutic targeting of toll-like receptors for infectious and inflammatory
diseases and cancer. Pharmacol Rev 61:177-197
This is to remind you of the TLR pathways. IL-12 is another inflammatory cytokine that is made when
PAMPS bind to the TLRs.
•
•
Signal 3 for differentiation of TH1 is the
best understood. See the upper half of
this somewhat confusing drawing.
The APC (still in the lymph node, so this
is likely a DC) secretes IL-12 for Signal 3,
when it engages TH cell (if this is what
the PRR/PAMP told it to do).
•
IL-12 causes a few TH cells to
differentiate into TH1 cells.
•
The TH1 cells then secrete IFN-g, which
stimulates the APC to secrete more IL-12,
increasing production of TH1. IFN-g also
inhibits formation of TH2 cells.( Other
effector cytokines such as Il-2 and TNF-b
are also secreted-we will talk about
those later).
•
IFN-g can also come from NK cells (and
CTL), in response to viral infection. The
macrophages and DC near the NK cells
will be exposed the IFN-g, and the APC
also will go to the lymph node, this time
secreting more IL-12. This helps make
TH1 which will help get rid of the virallyinfected cells (by helping Tc become CTL)
•
Signal 3 for differentiation of TH2 (lower
half of diagram):
•
IL-4 causes a few TH cells to differentiate
into TH2. The TH2 cells then secrete more
IL-4, which stimulates production of still
more TH2, and suppresses production of
TH1.
Where does the IL-4 come from? Not
necessarily the APC. Instead, other cells
(basophils, eosinophils, or mast cells)
can secrete this in response to “invasion” by
an allergen or helminth (worm) infection.
They also travel to the secondary lymph
tissue.
Note that there is cross-regulation, to make
sure one type of CD4 cell types
predominates
Embedded question: What is signal two in TH activation and why is it
necessary?
TREG can be made in thymus, during the process that gives
us naïve TH and TC cells. If TREG are made in the thymus, they
are called nTREG, for natural TREG. We will discuss that later
in the course
However, naïve TH cells can be directed to differentiate into
TREG cells as well. These are called induced TREG, or iTREG.
This usually happens in the gut, in the MALTs, because TREG
suppress our inflammatory immune response.
TREG help prevent us from attacking our gut microbiome.
Without TREG, the DC and macrophages in the MALT and
extracellular tissue in the gut would be responding to the
microbiome PAMPS and starting localized inflammatory
responses all the time.
One cytokine that can act as Signal 3 to make naïve TH
differentiate into iTREG is TGF-b. When naïve TH cells are
exposed to TGF- b, a transcription factor called FoxP3 is
turned on, and FoxP3 turns on genes that TREG cells use to do
their effector function job (which is to suppress the immune
system, and we’ll talk bout effector functions later in the
course).
Signal 3 for Treg
is TGF-b.
In our gut, our microbiome
(probiotic bacteria) send
signals (discussed in next
slide) to tell dendritic cells to
make IL-10, TGF-b, and
Retinoic Acid (RA). These act
as a Signal 3 that tells naïve
TH cells (called TH0 in this
picture) to differentiate into
iTREG.
Pathogenic bacteria (not part
of our microbiome) have
PAMPS that tell the dendritic
cells to secrete the Signal 3
cytokines that make naïve TH
cells differentiate into TH1, TH2,
and TH17 cells, which can all
increase the inflammatory
response.
The TREG cells can leave the
MALTs and go all around our
body, helping to control our
immune response. We will
talk about that more later.
If fed the correct stuff, like dietary fiber, the bacteria in the gut microbiome secrete things like small
chain fatty acids (SCFA). SCFA inhibit TLR signaling by sentinel cells, to reduce recruitment of immune
cells like neutrophils. This helps the good bacteria in our microbiome evade our immune system.
SCFA also induce dendritic cells to make TGF-b, IL-10, and Retinoic acid (RA). These three cytokines ac
as Signal 3 for iTreg, and also inhibit differentiation into TH1 and TH17 cells.
Anti-inflammatory diets give our microbiome bacteria the right food to make SCFA. Unfortunately, the
right food is more like green leafy vegetables and less like chocolate cake.
In the MALT, the signal 3 cytokines that cause activated TH to
differentiate into TREG can also come from other TREG cells.
This means that TREG can convert activated naïve TH cells into
iTREG cells. Since I am a nerd, this reminds me of the Borg,
which is why that picture is below. Don’t stress about it if you
don’t get it.
Now we know how naïve TH (CD4+) cells are activated
and how they differentiate into the effector cells (TH1,
TH2, TH17,TFH, and TREG) (we didn’t discuss TH17 or TFH
much, because we do have time limits). We will talk
about what the effector cells do later in the course.
Virus-infected DC
This happens in secondary lymph tissue.
How are naïve TC (CD8+) cells activated, and how do
they differentiate into CTLs (cytotoxic T lymphocytes)?
Il-2R
Il-2
The simplest way is for a Dendritic cell to become
infected by a virus at the site where the virus enters
the body (say, in the lungs, where someone inhaled
droplets containing SARS-CoV-2). We will come back
to how DC got this infection in a couple of slides.
The ribosomes in the infected DC will now start
making viral proteins. Some of the viral proteins will
be degraded by the Ub-proteasome system, and
presented on MHC I using the endogenous antigen
pathway.
The DC will go to the nearest lymph node (or other
secondary lymphoid tissue) and find Tc which, like TH,
are in the paracortex.
Virus-infected DC
Once near the Tc, the infected dendritic cell and the
Tc will interact (chemokines from DC attract Tc)
Signal 1 is still MHC-1/Ag-TCR/CD3, and MHC I-CD8.
Signal 2 is still B7-CD28.
The two signals combined activate NFAT, AP1, and
NFkB transcription factors to turn on the gene for IL-2
and IL-2 receptor. If you don’t have B7-CD28, only
NFAT is turned on, and you don’t get IL-2, you get
ANERGY. So far this is just like activation of TH cells.
IL-2 is secreted by the Tc cells, and it binds to the IL-2
receptor, causing the Tc to start proliferating.
Signal 3, which is not shown in the picture to the left,
is IL-12. IL-12 comes from the infected DC, and is
made when the viral PAMP activated a TLR . You can
see the pathway if you go to the earlier slide that has
all the TLR paths.
The activated Tc differentiates into a CTL. We will
talk about how CTLs work later in the course.
This is what one hopes will happen.
Sometimes, the Tc does not make enough IL-2 to drive
proliferation and survival of the Tc. It depends on what
the virus is, apparently. The problem seems to be
insufficient B7 on the DC-so signal two is too low.
In this case, the Tc needs help (cytokines) from a
nearby TH CD4 cell. This is usually a TH1 cell.
The CD4 cell is activated by the same DC as the Tc.
This can happen because:
1) The DC can phagocytose the virus as well
as be infected by the virus. This would allow
the viral proteins to be processed by the
exogenous pathway and presented on MHC
II.
2) The crossover pathway of Ag
presentation, which I told you to not worry
about. You can go back and look at it if you
want.
TH cells will bind to the same APC as the Tc (but will
bind to MHC II-Ag). They will bind whatever B7 is
there. It takes less signal 2 to activate TH cells than Tc
cells. Signal 2, in addition to turning on the IL-2 gene,
will turn on expression of CD40L, which binds to CD40
on the APC.
Binding to CD40 starts a signaling cascade that results
in the APC making more B7. It also causes the APC to
make 4-IBBL, which is a member of the TNF family and
is another co-stimulator of Tc cells. It binds to 4-IBB on
the Tc.
The extra co-stimulation, will cause the Tc to also turn
on the genes for IL-2 and IL-2R. This, combined with
the extra IL-2 from the TH cell, drives Tc proliferation
and survival.
IL-12 from the APC will still be signal 3 to differentiate
into CTL. It could also be signal 3 for the TH cells, and
cause them to become TH1 cells. TH1 can also help
activate Tc.
DC-SIGN
We have talked briefly about how viruses infect only certain cells because the bind to surface
protein receptors that are only expressed on certain cell types. For example, SAS-CoV-2
infects cells that have the ACE2 receptor on their surface.
So how can Dendritic cells be infected with viruses to present on MHC I?
It turns out that DC have sticky proteins on their surface, called DC-SIGNs. Most viruses can
bind to these and use this receptor to infect the DC. This allows DC to present most viral
antigens on MHC I, so that CTL that are specific to that virus can form.
The virus shown in this picture is HIV, and it also binds to other things. Don’t worry about that
right now.
Types of effector T cells
We have finally made T cells that can DO SOMETHING!! We’ll talk about what they
do to keep us healthy right after we activate some B cells.
Dendritic cells and macrophages are sentinel cells. Detect
pathogen type using TLR, NLR, RLR. Pick up antigen-process.
Able to present on MHC II.
Binding of PRR and activation of the signaling
path causes the Dendritic cells to migrate, carried
by the increased amount of fluid to the lymphatic
system.
They enter the lymph tissue (e.g. lymph node)
and the combined action of signaling molecules
from the TLR and from the lymph tissue cells
causes expression of the genes needed to activate
the TH cells (for example B7, called in this picture
CD80 and CD86, and increased MHC II.
INNATE
ADAPTIVE
Resident dendritic cells pick up (eat, phagocytosis)
the pathogen/antigen, and migrate using the
lymphatic system to a nearby lymph node.
Some of the pathogen also migrates with the lymph
on its own
Lymph fluid is increased by exudate that comes from
the innate response
In the lymph node, the
dendritic cell presents
antigen to naïve TH cells
to start the adaptive
response
B cells and resident DC or
Macrophage can pick up free antigen
Dendritic cells are one type of Antigen Presenting
Cells or APC.
APC’s “eat” antigens, and digest them in lysosomes,
and present them on MHC II.
Binding of PAMPs to PRRs on Dentritic cell surface
increases expression of MHC II. It also increases
expression of a cell surface protein dimer called B7
(aka CD80, CD86). This is part of the signal complex
that activates TH cells. It binds to a protein called
CD28 on the T cells. More detail in the future.
The PRR binding also causes the DC to secrete
cytokines that help direct TH cell differentiation into
the different types of TH effector cells (TH1, TH2, TFH,
TH17, Treg.
All this happens in secondary lymphatic
tissue, which includes lymph nodes, the
spleen, and Mucosal Associated Lymphatic
Tissue, or MALTS.
Lymphatic organs and tissues
•
Primary lymphatic tissue: where lymphocytes
develop from precursor cells
– Thymus: T cell development
– Bone Marrow (bursa in birds) : B cell
development
•
Secondary lymphatic tissue: Where
lymphocytes interact with antigen-each type of
tissue is specialized for the pathogen’s route of
entry
– Spleen: antigens in the blood
– Lymph nodes : antigens in the interstitial
fluid
– MALT: mucosal-associated lymphoid
tissue; antigens in the mucosa
– WHY?? to maximize chance of immune
cells interacting with antigen
• Lymphatic system
collects all the fluid that
leaks out of blood
vessels into the
interstitial fluid. It drains
all tissues. Collection is
into lymphatic vessels.
• The fluid is eventually
returned to the blood
through the thoracic
duct, which empties into
the left subclavian vein
near the heart.
• Lymph nodes are
located at junctions of
lymphatic vessels.
Embedded question: name three types of secondary lymphatic tissue, and say
what route of antigen entry they are linked to.
Lymph nodes
• Three “layers”, but with dynamic
movement of cells
• cortex-contains follicular dendritic
cells (FDC) and B-cells-organized
into primary and secondary follicles
• Paracortex: T-cells, fibroblast
reticular cells (FRC), dendritic cells
• medulla-differentiated plasma cells
Note: Follicular dendritic cells are not blood
cells, and do not differentiate from the
hematopoietic stem cell. They are more like
fibroblasts. These are not the DC we already
know and love
IN
Antigens/pathgens and
Dendritic cells come intot
he node through the
afferent vessels, in the
lymph.
OUT
Lymphocyte homing
•
Lymphocytes (T and B cells) enter the
nodes from the blood., by the process
of extravasation. It is similar to what
we learned for neutrophils; the
process uses cell adhesion molecules
on lymphocytes and blood vessels to
slow lymphocytes, then there is
activation which causes a change in
integrins, and then arrest/adhesion,
and extravasation.
In lymph nodes and some other
secondary lymph tissue, this happens
in specialized areas of post capillary
venules called HEV’s (high
endothelial venules). Cells in these
HEV’s have the correct cell adhesion
molecules
1 x 104 lymphocytes leave blood through
HEVs into a lymph node every
second.
Where do the chemokines come from?
FDC and FRC
Selectin is on the lymphocyte. Mucin-like CAM is on the HEV cell. This is different from neutrophils
B cell chemokine is CXCL3
T cell chemokines are CCL19, CCL21
Chemokines come from FCD, FRC in the nodes
•
•
•
•
•
After B cells go through the HEV, chemokines (CXCL13) from the FDC attract
them to the cortex by binding to the CXCR5 receptor on B cells.
In cortex, the primary follicles are loosely organized naïve B-cells and FDC
these B-cells are looking for antigen (remember, B cells can bind free antigen.)
They will not stay in this node for long (1 hour if not stimulated)
MRC are marginal reticular cells-they are part of the network (stroma)
supporting the node-they, and FDC, are thought to make conduits for antigen
flowing in with the lymph
T cells come in through the HEV, and migrate to the paracortex, guided by
chemokines (CCL19, CCL21) from the FRC . T cells have receptors for the
chemokines (CCR7). Dendritic cells also are recruited by these chemokines. T
cells crawl around the FRC network, interacting with dendritic cells that came
into the node with the lymph, and are attached to the FRC.
• Antigens in interstitial
fluid are picked up by
lymph, carried to
nearest lymph node
• antigen may have
been picked up,
processed by
dendritic cells, also
carried in by lymph
• Antigens (Ag) trickle through node
• Ag interact with B-cells in Primary
follicle; B-cells get primed (discuss in
detail later in the course)
• Ag get ingested by dendritic cells (in
tissue or in node), is presented on MHC
II.
• APC present to TH in paracortex
• If a TH is activated, it differentiates (TH1,
TH2, etc). TH and primed B cells move to
the periphery of the paracortex, where
the B cells can interact with the TH cells.
• B-cells will then proliferate and
differentiate into plasma cells and
memory cells, forming a secondary
follicle.
• plasma cells move to medulla, into
lymph (leave using the efferent
lymphatic vessel) and finally enter the
blood
Cortex
paracortex
• Secondary follicles are more
organized than primary
• They contain areas of dividing
B-cells and FDC in the
germinal center, surrounded
by densely packed
lymphocytes (B cells, memory
cells), called a mantle.
• Secondary follicles form after
antigenic stimulation
• Memory cells stay in contact
with FDC-will discuss more
later in course
Spleen- a filtering organ
•
•
•
•
•
•
There are two types of tissue in the
spleen-red pulp and white pulp.
Blood comes in through blood
vessels that empty out into sinuses
containing macrophages (which is
the red pulp)
The macrophages eat old/damaged
red blood cells. They also eat
opsonized bacteria bacteria in the
blood.
The blood could contain antigens. If
so, the macrophages will eat them
too. Dendritic cells can enter the
spleen this way too.
white pulp is the lymphoid tissue-B
and T cells.
In the white pulp, the T and B cells
are separated into two distinct
regions: the marginal zone, and the
peri-arteriolar lymphoid sheath
(PALS)
Macrophages and Dendritic Cells are in both red and while pulp. Macrophages recognize
dying RBC (changes in membrane lipids act as “eat me” signals), but this does not activate
the immune system, because they are “self”-no PAMPS. Macrophages can eat free antigen,
and can be Antigen presenting cell (APC) to T cells. Dendritic cells remain the most active
APC for naïve T cells.
•
Marginal zone:
–
–
•
PALS:
–
•
is between red pulp and PALS
contains B cells, macrophages
rich in T-cells, like paracortex of lymph nodes
There is not as clear a reticular network as is seen in lymph nodes, but T and B
cells coming in with the blood find their way to the correct place, using
chemokine signals. B cells form primary follicles, organized by chemokines
secreted by a splenic version of Follicular Dendritic Cells. T cells are recuited to
the PALS by other chemokines. DC go to the PALS to activate T cells, which
then move toward the B cells.
MALTS
mucosal associated lymphoid tissue
•
•
•
•
•
•
Found in all mucosal tissue (Gut-associated is
GALT; Bronchial is BALT, etc)
Can be well organized or very loosely
organized. The figure shows a Peyer’s patch,
which is well organized. Tonsils, adenoids,
appendix-all MALTS
MALTS protect mucosal tissue. Quite a lot of
pathogens enter through our respiratory and
gastrointestinal systems, so this is very
important to human health.
About 50% of lymphocytes in the body can be
found in MALTS
Lymph (carrying antigen and Dendritic cells)
also flows from intestinal lining and lung tissue
to nearby lymph nodes, which we have
already covered
SED is sub-epithelial dome, Dendritic cells
hang out there
follicle
T and B cells are segregated.
They get into MALTS using HEV (High Endothelial Venules),
like they do with lymph nodes. Leave blood using
extravasation (rolling, activation, arrest, migration).
The B cells are organized into follicles, using chemokines
and FDC. T cells are outside of the follicles, usually in the
spaces between follicles.
The antigen gets into the follicle from the lumen of the gut
by passing through specialized cells, called M cells, in the gut
epithelium. (marked (a) in picture). Dendritic cells hang out
near the M cells in the SED, and pick up the antigen
(Phagocytosis, uses PRR for PAMPS on pathogen. Antigen is
brought to lysosome, digested, and presented on MHC II.)
DC present antigen to TH cells (b) and activate them. They
differentiate to TH1 or TH2, depending on signal the DC gives
them (which depends on PAMPS).
B cells in follicles can also pick up antigen from M cells, and
process it. T cells can either activate a B cell in the same
patch of MALT, or go to another site (via lymph) to find a B
cell.
•
•
•
•
Specialized cells (M cells, or microfold cells) in the epithelial
cell layer transport antigen from the lumen of the gut to the
MALT.
M cells have “pockets”, where lymphocytes and DC can sit
in less organized MALT tissue.
M cells are specialized cells that pass antigen (undigested)
across epithelial layer
Both figures show essentially the same thing
•
B-cells in the MALT can be
activated by T cells, and form
secondary follicles where they
differentiate into plasma cells that
migrate along submucosa.
•
The plasma cells secrete IgA,
which is an immunoglobulin that
can be secreted into the lumen of
the gut. There IgA binds antigens
and prevents them from infecting
us.
We will discuss Ig A and other immunoglobulins later in the course.
In the lymph nodes (and MALTS, and spleen) the antigen is taken up by an Antigen Presenting Cell (APC).
APC process antigen and present it to TH cells on MHC II. The major APC are dendritic cells,
macrophages, and B cells. DC are the ones that are best at activating naïve TH cells. These three are
called Professional APC. Other cells types can be stimulated (for example, by inflammation) to be nonprofessional antigen presenting cells.
Professional APC are special for 4 reasons:
1) They can take up antigen by endocytosis or phagocytosis
2) The can express MHC II. Not all cell types can.
3) They express MHC II either all the time (constituitive) or when induced
4) They can express the co-stimulator B7.
To activate T cells, the APC must express both MHC II and co-stimulator B7. In
macrophages, TLR signaling can turn on (induce) MCH II signaling, and so can cytokines (IFNg) from TH1 cells.
Nonprofessional cells can be induced to express these, mostly when there is prolonged
inflammation (inflammatory cytokines, including IL-12)
All cell types are able to express MCH I to some extent, so any cell can process and
present antigen on MHC I to TC cells. However, not all cells go to the lymph node, so in the
lymph node, Dendritic cells are mostly the ones that present to Tc. DC can be infected with
most viruses, even though viruses generally have specificity to specific cell types. (For
example, coronavirus binds to ACE2 receptors, which are expressed by epithelial cells in
the lung, and to some extent, in the GI tract). Once Tc are activated in the secondary lymph
tissue (which is something we’ll go over later in the course), they differentiate into CTL, and
go out into the rest of the body, where the infected cell can present on MHC I (altered self)
to CTL and cause the CTL to kill the altered self cell.
Despite the label on this picture, the term “APC” generally refers to cells that present
antigens to TH on MHC II.
A brief summary:
Antigens from viruses are cytosolic. They are made when the host ribosomes in infected cells translate
viral proteins. Cytosolic pathogens are degraded by the Ubiquitin-proteasome system, and the
degradation products are displayed on MHC I. The presentation is by Dendritic cells to TC in the lymph
node, and by infected cells to CTL in the periphery. .
Viruses, cancer?
Antigen made on
host ribosome
Mycobacteria, Listeria
most bacteria
Antigen made on pathogen ribosome
A brief summary:
Antigens from both intracellular and extracellular bacteria are made on the pathogen ribosomes. They
get into Dendritic cells, macrophages, or B cells by phagocytosis or endocytosis. They are degraded by
lysosomes, and the degradation products are displayed on MHC II to TH in the lymph node.
Viruses, cancer?
Antigen made on
host ribosome
Mycobacteria, Listeria
most bacteria
Antigen made on pathogen ribosome
This is an overview of antigen
processing (degradation) and
presentation (attachment to MHC).
Endogenous antigens are made on host
ribosomes inside the host cell (e.g. viral
proteins). They are processed using the
endogenous pathway.
Exogenous antigens are made outside
the cell or in endocytotic vesicles, and
their synthesis does not use host
ribosomes (e.g. extracellular or
intracellular bacteria). They are
processed using the exogenous
pathway.
One interesting point is that it might be
possible for exogenous antigens to
“crossover” to the endogenous pathway,
so that MHC I could present a bacterial
(exogenous) peptide. We won’t really
discuss that, but its on the figure so I
mentioned it.
We’ll focus on the
endogenous path for MHC I
processing first.
The antigen is made by host
cell ribosomes
The MHC I peptides (there
are two, see the picture on
the right) are made on
ribosomes attached to the
Rough ER. The MHC I achain is an integral
membrane protein in the ER
membrane.
The b-chain is called b2microglobulin.
MHC I
The Ubiquitin-proteasome pathway degrades the non-self antigen. The
proteasome will also degrade normal (self) proteins. This happens in the cytosol.
Note that because the antigen is digested, epitopes that are internal on the
antigen are also going to be presented to T cells.
The antigen fragments have to bind the MHC I while it is still inside the cell (not on
the surface). The MHC I antigen binding site is on the a chain, which is inside the
the Rough ER, because that is where proteins destined for the Plasma membrane
are made. The antigen peptides are transported into the RER by a transporter
called TAP (Transporter associated with Antigen Processing). Energy for transport
is provided by ATP hydrolysis.
The newly synthesized MHC I a chain is an integral membrane protein in the RER
membrane. With help from the chaperone protein calnexin, it folds correctly and
associates with b2 microglobulin. (Chaperones are proteins that assist in folding and
assembly of other proteins). Calnexin is released, and another chaperone,
calreticulin, binds to the MHC I along with tapasin, which brings the MHC I close to the
TAP. ERp57 is a protein that first acts to stabilize the complex until MHC I binds a
peptide, and then causes release of the MHC I-Ag from the rest. The MHC I-Ag then
goes to the cell surface.. It won’t go if it does not have a peptide; it will get
degraded because the cell sees it as incompletely folded. Any protein digested by
the proteasome, including self peptides, will bind to MHC I.
MHC I
• MHC I protein has two peptides.
• The a peptide binds the
endogenous antigen peptide
• The b peptide, b2-microglobulin,
is structural.
• MHC I is not complete until it has
the a and b peptides joined
together, AND has bound a
peptide fragment.
• Incomplete proteins are not
transported out of the ER.
• If MHC I is on the surface, it
must be bound to a peptide.
• If the cell is not infected MHC I is
bound to a SELF peptide
• The Thymus removes most
developing Tc cells that do not
bind MHC, or which react with
MHC I bound to self peptide.
Binding the antigen fragment
changes the shape of the MHC
in the region that interacts with
T and NK cells.
For CTL to recognize infected cells, the infected cell must present non-self antigen on
MHC I. All of these viral evasion strategies decrease MHC I expression of viral
antigen, which allows the viruses to evade to immune system for a while. (However, as
we discussed earlier in the course, NK cells are more likely to kill cells with low MHC I.
But, without CTL recognition, the virus infection is unlikely to be removed).
https://www.youtube.com/watch?v=VPvCekgPwRI
This is a link to a pretty good video about MHC I
assembly and presentation.
https://www.youtube.com/watch?v=soWtpAO1Nr0
This is another one for MHC I
https://www.youtube.com/watch?v=TMnkihN6zVM
This is a link to a video about MCH II presentation.
We’ll focus on MHC II next. Remember,
this is happening after the antigen is
brought into the cell by phagocytosis or
endocytosis by APC.
In the Rough Endoplasmic Reticulum,
(RER), the MHC II peptide chains (see
next slide) are being synthesized. For
those of you who have had Cell Biology,
you will recall that proteins destined for the
surface of the cell are made in the RER.
Antigen fragments
MHC II
• The MHC II protein
contains two peptides, an
a and a b chain. Both are
made in the RER, and
they are joined together
in the RER.
• The antigen binds in a
cleft between the two
chains.
• Binding of antigen
changes the shape of the
MHC slightly, especially
in the region that interact
with T cells
Antigen binding cleft
Overview:
The MHC II travels in a vesicle to the
endosome/phagosome/lysosome
containing the digested antigen fragments.
The vesicle fuses to the lysosome.
The digested antigen fragments bind to the
MHC II and the MHC II-Ag complex gets
transported to the surface of the cell.
Antigen fragments
In the RER, the a and b chains of MHC II are made, and the two peptides are
assembled. No self proteins stick to the new MHC II on its way to the lysosome,
because the antigen binding site is blocked by a protein called the Invariant chain (in
one of the videos, it is called Ii). The invariant chain also helps with folding of the
MHC 2 chains, and with sending the MHC II on the correct transport path.
The path from the ER is to the Golgi, then to early endosome, late endosomes, and finally to a
lysosome, which contain the digested antigen fragments.
The proteases in the endosomes/lysosome gradually degrade the invariant chain, leaving a
small piece behind called CLIP. CLIP continues to block the antigen binding site.
Digested invariant chain
Embedded question: What happens to MHC I peptides if they can’t bind antigen?
What effect would this have on levels of Tc cells?
To remove CLIP, a special MHC II called HLA-DM (MHC II terminology will be
described later) is used. HLA-DM does not bind or present antigens-it lives in the
endosomes.
HLA-DM mediates the exchange of CLIP for a real antigen peptide. The real MHC IIAg goes to the cell surface. Another special MHC II called HLA-DO helps regulate the
exchange.
If MHC II is on the surface, it must be bound to a peptide.
If there is no pathogen present, MHC II is bound to a SELF peptide (but remember,
most cells don’t make MHC II, and those that do are induced to do so by PAMPS).
A self peptide is a normal cellular protein.
The Thymus removes developing TH cells that react with self MHC II or which do not
bind to MHC at all.
This is a summary slide that puts it all together.
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Both MHC I and MHC II are encoded by genes that are linked together on a single
stretch of DNA (stretching over 4 million bp) on chromosome 6 in humans. It is called
the HLA locus or the MHC locus (HLA stands for Human Leukocyte Antigen)
In the MHC/HLA locus, there are three classes of genes
Class I genes (pink) encode the a chain of MHC I proteins. Note that there are three
genes (HLA-A, HLA-B, and HLA-C) that encode the MHC I a chain. MHC I is
polygenic.
Class II genes (blue) encode the a and b chains of MHC II proteins. Note that there
are three gene pairs. MHC II is also polygenic
Class III genes encode other things, such as complement proteins, TNF
The gene for b2 microglobulin is not in this locus; it is on chromosome 15.
a and b chains
of MHC II
These encode the a
chain of MHC I
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Because there are multiple genes encoding the MHC I and II proteins, the MHC
genes/proteins are polygenic. All the genes (within the MHC I or II group) encode
similar proteins, but the proteins have slight sequence and structural differences.
All three genes/gene pairs in each group are expressed, and both copies of
chromosome 6 are expressed.
Within a population, there are many different variants of each of the genes for MHC
I a chain and for the two MHC II chains-the genes are polymorphic. So, even MHC
proteins encoded by the same gene type (e.g. HLA-A) can have slightly different
sequences and structures.
All the MHC genes are linked, and are inherited together as a single genetic
trait. Humans (diploid) have two copies of the locus, one from dad, one from mom.
Note that in the class two region, there are also genes for the TAP transporter, and
for subunits of the proteasome. In the class III region, there are genes for
complement proteins and for some cytokines, among other things.
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There are three main distinct genes for the
MHC I a peptide (polygenic), so human
express three slightly different a chains
encoded by each class I region.
The genes are called HLA-B, HLA-B, HLAC. Each gene is expressed.
Among humans, there are many variants
(alleles) of these genes (polymorphic). Your
parents are unlikely to have the same
alleles as each other, and you are unlikely
to have the same alleles on both your
chromosomes.
You are diploid, so you have 6 different
genes (One A from mom, one A from dad;
one B from mom, one B from dad, one C
from mom, one C from dad). You probably
have different alleles for each gene. Each
gene is expressed, so you probably have
6 different MHC I proteins on the surface
of your cells
You also have minor (non-classical) MHC 1
genes, which we won’t discuss here.
Currently, in humans, there are 6425
known HLA-A alleles, 7754 known HLAB alleles, and 6329 known HLA-C alleles.
This number constantly changes as more
people and more diverse populations are
examined.
There are 3 major gene pairs (polygenic)
for MHCII: DQ, DP, and DR. The A gene
encodes the a chain, and the B gene
encodes the b chain. DO and DM are
used for CLIP displacement.
The DP gene products pair with each
other, as do the DR and DQ products.
The final MHC II proteins is called HLADP, -DQ, or –DR. All 3 sets are
expressed.
Among humans, there are many variants
(allele) of each of the genes
(polymorphic). Your parents are unlikely
to have the same allele. So you probably
have 6 different versions of MHC II genes
expressed on APC.
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Within a species, the MHC genes are very polymorphic.
What this means is that in a population, there are a bunch of slightly different nucleotide
sequences encoding the DPa1 gene, and so on. The different genes are called alleles.
There are a lot of different alleles, that can differ in DNA sequence by as much as 10%, and may
differ in as many as 20 aa.
You have possibly two alleles for each gene; one allele from mom, and one allele from dad. It is
possible that mom and dad might have some of the same alleles, but it is extremely unlikely that
many of their alleles are the same, unless they are related.
You inherit the ENTIRE HLA locus (all 4 million plus bp) as a linked unit.
Your set of MHC I and MHC II genes/proteins is pretty rare-hard to find someone who matches
you.
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3621
1674
233
1968
279
29
Why so polymorphic? May help bind a diverse set of antigen peptides. Most of the
variation among different alleles is in the peptide binding cleft.
Since antigens need to be presented, they have to bind to MHC. If all the MHC are the
same, then some peptides might not be able to be presented to T cells, and there
would be no response.
One of the problems with inbreeding-genetic diversity. First noted in inbred mouse
strains-a failure to respond to some antigens. Taken into consideration when
managing populations of endangered species.
Each identified allele has a number (e.g. DP-A60).
Your HLA haplotype is the entire set of your alleles in the HLA-region.
Your HLA-region on one of your chromosome 6’s will be identical in all
alleles to the region on one of your mother’s two chromosome 6s. The
HLA region on your other chromosome 6 will be identical to one of your
fathers two HLA-regions.
You have a ¼ chance of having the same HLA haplotype as one of your
siblings.
During T cell development, T cells that bind tightly to the host set of MHCs are
removed to prevent auto-immune diseases. But, MHCs encoded by heterologous
alleles have a slightly different shape than yours, so your T cells see tissues from
other people as altered self.
If you get a tissue transplant, your T cells will probably attack the transplant unless the
MHCs from donor and recipient match.
For tissue/organ transplants, usually they look at HLA-A, HLA-B alleles for MHC I
(which is on all cell types) ,
And HLA-DR alleles for MHC II (which is only on APC).
Try to get the best match. Also need to match blood type.
Here is an example of what tissue typing might look like.
Your best chance to get a match is a sibling. In the general population, members
of the same ethnic/racial group are more likely to be matches.
Can lead to disparities in access to transplanted organs.
Antigens
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By definition, antigens are molecules that bind to
antibodies (or Immunoglobulin)
However, now we tend to think of them as things
that bind to antibodies, or T Cell Receptors
(TCR) or immunoglobulins on surface of B cells
(mIg)
If antigens elicit an immune response they are
considered to be immunogens.
Some very small molecules can bind to
antibodies but do not raise an immune responsethese are called haptens.
The part of the antigen that physically makes
contact with the antibody protein (or TCR) is
called the epitope. Antigens can have multiple
copies of the same epitope, as well as a lot of
different epitopes.
We will go over how the TCR or Ig bind the
epitopes later in the course.
The epitope of an antigen binds
to the ends of the surface
immunoglobulin on a B cell.
The binding is specific.
All the surface immunoglobulins
on a single B cell bind to the
same epitope.
Every B cell has slightly different
antigen-binding sites on the
surface immunoglobulins, so
every B cell binds a different
epitope.
• Epitopes are parts of antigens.
• most antigens have lots of
different epitopes-the protein
pictured on the right is the
surface glycoprotein of Ebola
Virus. Shown are 4 different
epitopes, each of which are part
of a different functional region
(mechanism of action, or MoA) of
the protein.
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Since most pathogens have more than
one copy of their surface proteins, the
surface of a pathogen will have multiple
copies of each epitope. This becomes
more relevant when we get to how B cells
are activated.
This is a picture of the SARS-CoV-1 spike protein (the one that emerged in 2003.)
The stuff marked in red are epitopes that are genetically identical (same aa
sequence) to ones on the spike protein of the current virus, SARS-CoV-2.
Knowing B cell epitopes, and also knowing the Mechanism of Action of those
epitopes (for example, the epitopes in the region where Spike interacts with the host
cell receptor) help with vaccine development and evaluation.
You want to generate antibodies that block the epitopes involved in host binding,
since this will prevent infection.
doi: https://doi.org/10.1101/2020.02.19.955484
Are the mutations in epitopes
with important MoA? Or in
epitopes that are more likely to
bind to B cells and generate
protective antibodies?
Although all epitopes have a specific conformation, the amino acids contribution to
that conformation can be next to each other (sequential/linear) or folded together
as part of the proteins tertiary structure (conformational). This also matters when
looking at the mutations of a pathogen, to see if they are going to prevent an
immune response
Pathogen: SARS-CoV-2
Antigen: Spike protein
Epitope: S1-RBD, the amino acids in Spike that bind to the host receptor ACE2.
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For B-cell antigens: The antigens are usually undigested, and on the surface of
a pathogen (unless it is an isolated protein). The epitopes are usually on the
surface of the antigen..
For TH-cell antigens: proteins (or entire pathogens) are picked up by antigen
presenting cells, and the proteins are digested in lysosomes. Protein
fragments, containing the epitopes, are presented on the surface of the APC
by MHC II. Because TH cells interact with digested antigen (mostly proteins),
the epitopes can either be on the surface of the antigen, or be internal amino
acids. The presented epitopes are small fragments, and they usually are
sequential
Epitopes recognized by T cells must be presented on MHC I or MHC II
Antigen binding to B cells; no presentation needed
Antigen binding to T cells requires that an Antigen Processing Cell (APC) digest an
internalized pathogen, and present it on MHC.
For TH, MHC II does the presenting. For TC, MHC I does the presenting.
MHC II binds antigen fragments that come into the cell from the outside, and are digested in
lysosomes. Only a few cell types can express MHC II.
MHC I binds antigen fragments that were made in the cell, and digested by the ubiquitinproteasome
For presenting antigen on MHC I.
All cell types are able to express MCH I to some extent, so any cell can process and
present antigen on MHC I to TC cells. However, not all cells go to the lymph node, so in the
lymph node, Dendritic cells are mostly the ones that present to Tc. DC can be infected with
most viruses, even though viruses generally have specificity to specific cell types. (For
example, coronavirus binds to ACE2 receptors, which are expressed by epithelial cells in
the lung, and to some extent, other tissues). Once Tc are activated in the secondary lymph
tissue (which is something we’ll go over later in the course), they differentiate into CTL, and
go out into the rest of the body, where the infected cell can present on MHC I (altered self)
to CTL and cause the CTL to kill the altered self cell.
Antigens are either picked up at the site of infection by sentinel cells, or they flow with the lymph
to the lymph nodes (or other similar tissue) and are is taken up by an Antigen Presenting Cell
(APC) in the node. APC process antigen and present it to TH cells on MHC II. The major APC
are dendritic cells, macrophages, and B cells. DC are the ones that are best at activating naïve
TH cells. These three are called Professional APC. Other cells types can be stimulated (for
example, by inflammation) to be non-professional antigen presenting cells.
P rofes s ional AP C are s pecial for 4 reas ons :
1) T hey can take up antigen by endocytos is or phagocytos is
2) T he can expres s MHC II. Not all cell types can.
3) T hey expres s MHC II either all the time (cons tituitive) or when induced
4) T hey can expres s the co-s timulator B 7.
Embedded question: name four features that allow cells to be professional antigen presenting
cells
To activate T cells, the APC must express both MHC II and co-stimulator B7. In
macrophages, TLR signaling can turn on (induce) MCH II and B7 expression, and so can
cytokines (IFN-g) from TH1 cells. It’s part of macrophage activation
Nonprofessional cells can be induced to express MHC II and B7, mostly when there is
prolonged inflammation (inflammatory cytokines, including IL-12)
The external TLRs, that recognize bacterial PAMPS are not shown, but also activate
the NF-kB and MAPK pathways, turning on CD80/86 (B7) and MHC II.
A brief summary:
Antigens from both intracellular and extracellular bacteria are made on the pathogen’s
ribosomes. They get into Dendritic cells, macrophages, or B cells by phagocytosis or
endocytosis. They are degraded by lysosomes, and the degradation products containing
epitopes are displayed on MHC II to TH in the lymph node.
Viruses, cancer? Mycobacteria, Listeria
Antigen made on
host ribosome
most bacteria
Antigen made on pathogen ribosome
A brief summary:
Antigens from viruses are cytosolic. They are made when the host ribosomes in infected cells
translate viral proteins. Cytosolic pathogens are degraded by the Ubiquitin-proteasome
system, and the degradation products containing epitopes are displayed on MHC I. The
presentation is by Dendritic cells to TC in the lymph node, and by infected cells to CTL in the
periphery. .
Viruses, cancer?
Antigen made on
host ribosome
Mycobacteria, Listeria
most bacteria
Antigen made on pathogen ribosome
Our next infectious agents are helminths
(gastrointestinal nemotodes, or worms).
Worms are BIG, relative to immune cells. Can’t
phagocytose them.
Helminth larva infect by passing through the
intestinal epithelial cell lining, into the submucosa. The adult worms generally stay
outside, unless the cell lining is damaged. So
need to try to expel them, and to strengthen the
mucosal barrier.
The worms have PAMPS, and cause tissue
damage, so there are also danger signals.
Mucosal Immunology (2018) 11, 304-315;
doi:10.1038/mi.2017.113
The epithelial cells of the intestine may detect
some PAMPS, and DAMPS, and secrete
cytokines that help strengthen the mucosal
barrier.
Dendritic cells pick up worm antigens,
mostly from infecting larvae, and present
to naïve TH in MALT or nearby lymph
node. TH2 cells are induced because this
is what the PAMPS in the worm are
directing. TH2 cells can direct B cells to
become IgE secreting plasma cells. TH2
leave the node and go to the infected
mucosa.
TH2 cytokines can activate macrophages
(process is called alternative activation
since it is TH2 and not TH1 doing it)-these
macrophages secrete toxic material,
increase inflammation, bringing in
neutrophils. The neutrophils try to
phagocytose the larvae.
Mucosal Immunology (2018) 11, 304-315;
doi:10.1038/mi.2017.113
The plasma cells that differentiated produce IgE. The IgE binds to Fc
receptors on the surface of mast cells, eosinophiles, basophils,
macrophages, and neutrophils, which are located in the sub-mucosa.
When the cells with bound IgE encounter adult worm antigens that the
IgE binds to, the cells release cytokines to bring in eosinophils and more
neutrophils, and also release stuff to increase mucus production, and
enzymes to digest adhesions between the worm and the intestinal lining.
The epithelial cells are also induced to multiply, thickening the barrier.
There is also increased muscle contraction, and this should help expel
the worm.
If the worms have penetrated the intestine, IgE bound to the worm may
activate complement, which can also bind to mast cells and cause
degranulation.
C3b and IgE bound to larva can act as opsonins and bind to neutrophils
which can try to phagocytose the larva, and/or release toxic mediators.
Eosinophils can be activated by IgE bound to the worm, or by factors
released by mast cells. The eosinophils release the contents of their
granules, which are toxic (anti-helminth peptides, reactive oxygen
species, other toxic stuff)
TH2 cells activate macrophages-phagocytosis, release of enzymes and
toxins in granules.
IL-4
TH2
This response will hopefully expel or kill any
adult worms and will kill larvae so the
infection does not get worse.
Luckily, there are also effective anti-helminth
drugs, but there is always the risk of reinfection or resistance.
IL-4
TH2
Immune response to viruses.
The recognition and proliferation/differentiation stages happen in the secondary
lymphoid tissue, and we have already covered it.
Antigen is presented on MHC I. Signal 3 produces CTLs
TH1 cells might be needed during the stimulation phase to generate sufficient IL-2
and co-stimulation to activate Tc.
• Once the CTL differentiate in the
secondary lymphoid tissue, they go off
into the rest of the body (through the
blood) and look for the altered self cells
that will present the specific antigen to
the T cell Receptor on the CTL.
Remember, each CTL will recognize only a
single epitope, so the action of the CTLs
is extremely specific.
• As it roams the tissues in the body, the
CTL non-specifically and weakly binds to
cells using cell adhesion molecules.
• Remember, all nucleated cells present
peptides generated using the Ubproteasome on MHC I. Normal cells
present self-antigens. Those will not be
recognized by any CTL TCR. If there is no
specific interaction through TCR, the CTL
will move on.
Infected or cancer cells present non-self antigens on MHC I. These get recognized by the
TCR, which binds to MHC I/Ag. CD8 binds to MHC I. CD8 and CD3 send a signal cascade in
side the cell.
Once the TCR makes a specific contact with MHC I/Ag, the CTL will use other Cell Adhesion
molecules to make bind tighter to the target cell. This is called conjugate formation, and it
is there to prevent the CTL from affecting any nearby cells. It also sends signals inside the
CTL.
The signals in the CTL cause rearrangement of the cytoskeleton, and granules containing
cytotoxic material is brought to the part of the CTL that is next to the target cell.
The granules move to the CTL surface, and merge with the plasma membrane, releasing the
toxic stuff.
All the cell-cell contacts between the CTL and target cell are then broken, and the CTL
moves on to the next target. The target cells dies by apoptosis.
Some nice pictures of the
CTL attacking the target
cell.
This is what is in the granules, and it’s the same as with NK cells.
Drawing of how granules fuse to plasma membrane, releasing perforin which
then forms pores in the membrane of the target cell. Perforin has homology
to C9 of the complement proteins.
An EM picture of what the pores look like
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Perforation alone does not kill the cells,
even though water should be able to
get in.
Perforation lets in granzymes,
particularly granzyme B
Granzyme B can also get in by binding
to a mannose-6-phosphate receptor,
and endocytosis.
The granzyme activates caspase 8,
which activates caspase 3, which
cleaves cellular components and causes
apoptosis.
CTL also have FasL on their surface, a
protein that that binds to the Fas
receptor, activating the death pathway
by yet another way.
The killing is neat and clean. The cells
don’t blow up, releasing their contents
all around. Remember, if the cells blew
up, there would be DAMPS and
alarmins, and this would activate TLR
and the inflammasome. With apoptosis,
there is little innocent bystander
damage, and little inflammation.
Macrophages and other phagocytes
come around and clean up the cellular
fragments left after apoptosis.
Once CTL are made, they eventually need to be controlled. This is done using surface
proteins similar to CD28 (which binds to B7, this is signal 2), except the action is
inhibitory, not stimulatory. One of the inhibitory proteins is PD-1, which binds to PD-L1
or PD-L2 on the non-self cell. This will block the signaling from the TCR/CD3 so the CTL
is not triggered to release its granules. In the picture below, this is called inhibition of
effector function.
PD-1 is made as part of CTL differentiation.
The PD-1 response is considered an
immunological checkpoint, since it
controls the immune system. It
helps us from having autoimmune
responses.
However, tumor cells in many
human cancers express high levels
of PD-L1 or PD-L2 (PD-ligand 1 or
2). This prevents our CTL from
removing the non-self tumor cells.
Immunotherapy, in the form of PD-1 blockers, is a newer from of cancer
treatment that in some cases is extremely effective. Cells from a tumor can
be tested to see if they express high levels of PD-L1/2, in which case this
could be a good treatment. Many of the new drugs are designed
monoclonal antibodies, which work by the antibody effector function of
neutralization.
This pretty much finishes up how our immune system responds to viral
infections, and tries to deal with cancer. Don’t forget the innate response,
with the NK cells and all.
Embedded question: name two ways TREG suppress immune responses
Finally, what do TREG do? In general they suppress the immune response by
preventing other types of TH cells from working.
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One way TREG cells work is by preventing
Signal 2 to in TH cell activation.
Remember, CD28 binding to B7.1, B7.2 is
required for activation.
B7.1,2 can also bind to a T cell surface
protein called CTLA-4. CTLA-4 is similar
to CD28, but is inhibitory,not stimulatory.
CTLA-4 is on TREG cells. It is one of the
genes turned on by FoxP3.
CTLA-4 binds B7 about 20 times more
strongly than CD28 does.
Binding to CTLA-4 is inhibitory,
because it competes with, and inhibits
binding to CD28. CTLA does not interact
with the same signaling path as CD28.
By preventing the CD28-B7 interaction,
TREG can induce ANERGY in TH1 or TH2
cells (GRAIL gets turned on instead of IL2 and IL-2R(CD25).
This is what
is affected
on the TH
cell
Nature Reviews Immunology 4, 841-855 (November
2004)
• TREG also stop TH cells from responding
to IL-2, if the TH cells got past the CTLA4 inhibition of B7.
• Normally, when B7 binds to CD28, IL-2
and IL-2R are made by the TH cell. IL-2
binds to the IL-2 receptor (CD25). This
starts a signaling path that causes the T
cells to enter the cell cycle and to
proliferate and begin differentiation into
effector cells or memory cells.
• IL-2 is also a survival signal, so if there
is no IL-2, T cells die
• In addition to CTLA, TREG cells have a
lot of CD25 (IL-2R) on their surface
(CD25 is another gene activated by
FoxP3). So they can titrate all of the
secreted IL-2,preventing the naïve T cell
from getting any. The naïve T cells die
from lack of IL-2 activation (and
immune system is suppressed).
TREG cells inhibit the immune system by:
• Depleting the local area of IL-2 by making a lot of CD25/IL-2R. Loss of
survival/proliferation signal causes the TH cells to die.
• Producing TGF-b which converts TH into TREG.
• Producing IL-10 which decreases expression of MHC and B7 on APC, and
decreases the production of IL-12 and IL-23, which decreases TH1 and TH17
differentiation cytokines (signal 3). So TREG inhibit APC from activating TH
cells
• Inhibit co-stimulation by using CTLA to inhibit B7 from binding to CD28. Will
result in anergy and death.
Converted to TREG
TFH?
TH1
IgA is the most prevalent Ig
produced in the gut, where
many TREG are.
Treg?
TH2
TH1,H2
We finally have antibody producing plasma cells! Some of the plasma cells are going to
circulate for a while, secreting antibodies to get rid of the infection, if the innate system hasn’t
already done it. Some will go to the bone marrow, where they live a long time, and produce
protective antibodies.
Plasma cells made in the MALT tend to stay there, and secrete IgA into the mucosa.
How do antibodies help get rid of infections? What type of infections???
Antibodies have 3 major effector
functions that help us fight disease:
1) Neutralization
2) Opsonization
3) Complement Activation
There are a few other functions that
we will hit in a few slides.
Neutralizing antibodies prevent the
antigen/pathogen from binding to a
cell’s surface. For example, an
antibody that bound to the SARSCoV-2 spike protein could
neutralize it, preventing it from
binding to the ACE2 receptor on
lung cells.
We have already learned about
opsonization and complement
activation (classical pathways was
activated by antibodies), and you
will see it represented in pictures
here.
More about neutralization:
In addition to binding to blocking
antigens from binding to a cell surface,
neutralizing antibodies can also bind
bacterial toxins and prevent them from
binding to cell receptors.
This is how the vaccine against diphtheria
and tetanus works. You get injected with
inactivated toxoid proteins and your body
builds antibodies that will neutralize the
toxin if you ever get infected with the
bacteria (Clostridium tetani,
Corynebacteria diphteriae).
Once the antigen is bound to neutralizing
antibodies, the stem (Fc) regions of the
antibodies can bind to Fc receptors on
macrophages, and the antigen is
destroyed in the lysosome. Opsonization
is similar, except a microbe is coated and
ingested, not just an antigen.
Neutralization also prevents toxins and pathogens from entering the body through
the mucosal layer. The Ig types that usually does this is IgA.
Neutralization 3 ways
Prevent a toxin from
binding to a receptor
Prevent a
virus from
binding to a
cell surface
receptor
Prevent a bacteria from binding to a cell’s surface
An effector function sort of related to opsonization is the ability of
antibodies to clear antigens complexes from blood or other places
in the body. This is related to complement, and is one of the
functions of complement we learned about earlier in the course.
Ab can bind soluble antigens, and form a larger complex where
the Ag cross-links multiple Ab together. In mucosal tissue, (lumen
of gut, for example) this will result in elimination.
In the blood, there is no obvious exit route for elimination.
However, the close spacing of the Ig Fc regions activates
complement via the classical pathway, and C3b is made. C3b lands
on the antigen/antibody complex.
The C3b can bind Complement Receptors (CRs)on red blood cells
or on phagocytes. The Fc portion of the Ab can bind Fc receptors
on macrophages in the spleen, which remove the complex and the
RBC.
Immune complexes can also cause problems (Type 2 and 3
hypersensitivity diseases).
So clearance of immune complexes is a antibody effector function.
Type III hypersensitivity-antibody complex disease
Immune complexes can be a problem-causing
inflammation. This is called Type III hypersensitivity.
It is one of the major pathologies found with some
auto-immune diseases. Example-Lupus,
Rheumatoid arthritis.
In Lupus, apoptosis leads to generation of
antibodies that react against self nuclear proteins
(this shouldn’t happen). Since we have a lot of self
proteins, there is a ton of antigen binding to the
auto-antibodies.
The antibodies make an immune complex, which
should get cleared.
But, if there is too much complex, the normal
methods of clearing them don’t work. The
complexes then bind to blood vessel walls, and
activate complement there, which causes
inflammation. They also can bind to the basement
membranes in the kidney glomerulus (filtering
tissue) and activate complement there too.
Inflammation. Results in kidney damage.
In RA, the immune complexes are deposited on
joint surfaces
•
When immune complexes are localized
by having antigens concentrated
intradermally, the type III
hypersensitivity reaction is the Arthus
reaction
•
It can happen with a booster shot of a
vaccine (when there are already
circulating antigens).
•
Mosquito bites are also an example of
the Arthus reaction
Other effector functions of antibodies:
• Antibody-dependent cell mediated cytotoxicity (ADCC). This involves antibodies
binding to non-self antigens on a host cell surface (like proteins produced as a
consequence of a virus infection, or cancer) or to a pathogen surface, such as a
helminth (worm). Effector cells (like NK cells, eosinophils) bind to the Fc portion
of the bound antibodies, and if this causes clustering of the Fc receptors, it starts a
signaling cascade that causes release of granules from the effector cell. Above, the
effector cell is an NK cell, which releases granzymes and perforins to kill the cell
by apoptosis, as we learned in the presentation about the innate response to viruses.
The effector cell could also be an eosinophil, which releases toxic and
inflammatory compounds.
• Mast cell sensitization. In this case,
an allergen or pathogen (again, this
happens with helminths) causes
production of antibodies (usually
IgE) that bind to Fc receptors on the
surface of mast cells. When the
sensitized mast cells encounter any
copies of the antigen in the body,
the bound immunoglobulins bind
the antigen, and this causes
clustering of the Fc receptors. This
sends a signal to the mast cell to
release granules containing
histamine and other inflammatory
substances.
This is the basis of the allergy
response.
The response can be local, or systemic.
If the response is systemic, (antigen gets into your blood) then you can get anaphylaxis
Engagement question: Name 5 effector functions of antibodies
IgE binds to mast cells
Each immunoglobulin class is suited for specific effector functions. The table
above summarizes which Ig are good at which functions. Remember, it is the Fc portion that
differs among the different Ig types.
Note that IgA is good at neutralization, not so good for ADCC. This is the Ig type made in
MALTs and secreted into the lumen of the intestines. Its job there is to prevent pathogens
from getting into the body through the GI tract.
IgGs (there are subclasses of IgG) are found in blood serum and are included in the exudate
that leaves the blood as part of the innate response to being jabbed with a pointy stick. They
are very good at opsonization and complement activation.
Our next infectious agents are helminths
(gastrointestinal nemotodes, or worms).
Worms are BIG, relative to immune cells. Can’t
phagocytose them.
Helminth larva infect by passing through the
intestinal epithelial cell lining, into the submucosa. The adult worms generally stay
outside, unless the cell lining is damaged. So
need to try to expel them, and to strengthen the
mucosal barrier.
The worms have PAMPS, and cause tissue
damage, so there are also danger signals.
Mucosal Immunology (2018) 11, 304-315;
doi:10.1038/mi.2017.113
The epithelial cells of the intestine may detect
some PAMPS, and DAMPS, and secrete
cytokines that help strengthen the mucosal
barrier.
Dendritic cells pick up worm antigens,
mostly from infecting larvae, and present
to naïve TH in MALT or nearby lymph
node. TH2 cells are induced because this
is what the PAMPS in the worm are
directing. TH2 cells can direct B cells to
become IgE secreting plasma cells. TH2
leave the node and go to the infected
mucosa.
TH2 cytokines can activate macrophages
(process is called alternative activation
since it is TH2 and not TH1 doing it)-these
macrophages secrete toxic material,
increase inflammation, bringing in
neutrophils. The neutrophils try to
phagocytose the larvae.
Mucosal Immunology (2018) 11, 304-315;
doi:10.1038/mi.2017.113
The plasma cells that differentiated produce IgE. The IgE binds to Fc
receptors on the surface of mast cells, eosinophiles, basophils,
macrophages, and neutrophils, which are located in the sub-mucosa.
When the cells with bound IgE encounter adult worm antigens that the
IgE binds to, the cells release cytokines to bring in eosinophils and more
neutrophils, and also release stuff to increase mucus production, and
enzymes to digest adhesions between the worm and the intestinal lining.
The epithelial cells are also induced to multiply, thickening the barrier.
There is also increased muscle contraction, and this should help expel
the worm.
If the worms have penetrated the intestine, IgE bound to the worm may
activate complement, which can also bind to mast cells and cause
degranulation.
C3b and IgE bound to larva can act as opsonins and bind to neutrophils
which can try to phagocytose the larva, and/or release toxic mediators.
Eosinophils can be activated by IgE bound to the worm, or by factors
released by mast cells. The eosinophils release the contents of their
granules, which are toxic (anti-helminth peptides, reactive oxygen
species, other toxic stuff)
TH2 cells activate macrophages-phagocytosis, release of enzymes and
toxins in granules.
IL-4
TH2
This response will hopefully expel or kill any
adult worms and will kill larvae so the
infection does not get worse.
Luckily, there are also effective anti-helminth
drugs, but there is always the risk of reinfection or resistance.
IL-4
TH2
Immune response to viruses.
The recognition and proliferation/differentiation stages happen in the secondary
lymphoid tissue, and we have already covered it.
Antigen is presented on MHC I. Signal 3 produces CTLs
TH1 cells might be needed during the stimulation phase to generate sufficient IL-2
and co-stimulation to activate Tc.
• Once the CTL differentiate in the
secondary lymphoid tissue, they go off
into the rest of the body (through the
blood) and look for the altered self cells
that will present the specific antigen to
the T cell Receptor on the CTL.
Remember, each CTL will recognize only a
single epitope, so the action of the CTLs
is extremely specific.
• As it roams the tissues in the body, the
CTL non-specifically and weakly binds to
cells using cell adhesion molecules.
• Remember, all nucleated cells present
peptides generated using the Ubproteasome on MHC I. Normal cells
present self-antigens. Those will not be
recognized by any CTL TCR. If there is no
specific interaction through TCR, the CTL
will move on.
Infected or cancer cells present non-self antigens on MHC I. These get recognized by the
TCR, which binds to MHC I/Ag. CD8 binds to MHC I. CD8 and CD3 send a signal cascade in
side the cell.
Once the TCR makes a specific contact with MHC I/Ag, the CTL will use other Cell Adhesion
molecules to make bind tighter to the target cell. This is called conjugate formation, and it
is there to prevent the CTL from affecting any nearby cells. It also sends signals inside the
CTL.
The signals in the CTL cause rearrangement of the cytoskeleton, and granules containing
cytotoxic material is brought to the part of the CTL that is next to the target cell.
The granules move to the CTL surface, and merge with the plasma membrane, releasing the
toxic stuff.
All the cell-cell contacts between the CTL and target cell are then broken, and the CTL
moves on to the next target. The target cells dies by apoptosis.
Some nice pictures of the
CTL attacking the target
cell.
This is what is in the granules, and it’s the same as with NK cells.
Drawing of how granules fuse to plasma membrane, releasing perforin which
then forms pores in the membrane of the target cell. Perforin has homology
to C9 of the complement proteins.
An EM picture of what the pores look like
•
•
•
•
•
•
•
Perforation alone does not kill the cells,
even though water should be able to
get in.
Perforation lets in granzymes,
particularly granzyme B
Granzyme B can also get in by binding
to a mannose-6-phosphate receptor,
and endocytosis.
The granzyme activates caspase 8,
which activates caspase 3, which
cleaves cellular components and causes
apoptosis.
CTL also have FasL on their surface, a
protein that that binds to the Fas
receptor, activating the death pathway
by yet another way.
The killing is neat and clean. The cells
don’t blow up, releasing their contents
all around. Remember, if the cells blew
up, there would be DAMPS and
alarmins, and this would activate TLR
and the inflammasome. With apoptosis,
there is little innocent bystander
damage, and little inflammation.
Macrophages and other phagocytes
come around and clean up the cellular
fragments left after apoptosis.
Once CTL are made, they eventually need to be controlled. This is done using surface
proteins similar to CD28 (which binds to B7, this is signal 2), except the action is
inhibitory, not stimulatory. One of the inhibitory proteins is PD-1, which binds to PD-L1
or PD-L2 on the non-self cell. This will block the signaling from the TCR/CD3 so the CTL
is not triggered to release its granules. In the picture below, this is called inhibition of
effector function.
PD-1 is made as part of CTL differentiation.
The PD-1 response is considered an
immunological checkpoint, since it
controls the immune system. It
helps us from having autoimmune
responses.
However, tumor cells in many
human cancers express high levels
of PD-L1 or PD-L2 (PD-ligand 1 or
2). This prevents our CTL from
removing the non-self tumor cells.
Immunotherapy, in the form of PD-1 blockers, is a newer from of cancer
treatment that in some cases is extremely effective. Cells from a tumor can
be tested to see if they express high levels of PD-L1/2, in which case this
could be a good treatment. Many of the new drugs are designed
monoclonal antibodies, which work by the antibody effector function of
neutralization.
This pretty much finishes up how our immune system responds to viral
infections, and tries to deal with cancer. Don’t forget the innate response,
with the NK cells and all.
Embedded question: name two ways TREG suppress immune responses
Finally, what do TREG do? In general they suppress the immune response by
preventing other types of TH cells from working.
•
•
•
•
•
•
One way TREG cells work is by preventing
Signal 2 to in TH cell activation.
Remember, CD28 binding to B7.1, B7.2 is
required for activation.
B7.1,2 can also bind to a T cell surface
protein called CTLA-4. CTLA-4 is similar
to CD28, but is inhibitory,not stimulatory.
CTLA-4 is on TREG cells. It is one of the
genes turned on by FoxP3.
CTLA-4 binds B7 about 20 times more
strongly than CD28 does.
Binding to CTLA-4 is inhibitory,
because it competes with, and inhibits
binding to CD28. CTLA does not interact
with the same signaling path as CD28.
By preventing the CD28-B7 interaction,
TREG can induce ANERGY in TH1 or TH2
cells (GRAIL gets turned on instead of IL2 and IL-2R(CD25).
This is what
is affected
on the TH
cell
Nature Reviews Immunology 4, 841-855 (November
2004)
• TREG also stop TH cells from responding
to IL-2, if the TH cells got past the CTLA4 inhibition of B7.
• Normally, when B7 binds to CD28, IL-2
and IL-2R are made by the TH cell. IL-2
binds to the IL-2 receptor (CD25). This
starts a signaling path that causes the T
cells to enter the cell cycle and to
proliferate and begin differentiation into
effector cells or memory cells.
• IL-2 is also a survival signal, so if there
is no IL-2, T cells die
• In addition to CTLA, TREG cells have a
lot of CD25 (IL-2R) on their surface
(CD25 is another gene activated by
FoxP3). So they can titrate all of the
secreted IL-2,preventing the naïve T cell
from getting any. The naïve T cells die
from lack of IL-2 activation (and
immune system is suppressed).
TREG cells inhibit the immune system by:
• Depleting the local area of IL-2 by making a lot of CD25/IL-2R. Loss of
survival/proliferation signal causes the TH cells to die.
• Producing TGF-b which converts TH into TREG.
• Producing IL-10 which decreases expression of MHC and B7 on APC, and
decreases the production of IL-12 and IL-23, which decreases TH1 and TH17
differentiation cytokines (signal 3). So TREG inhibit APC from activating TH
cells
• Inhibit co-stimulation by using CTLA to inhibit B7 from binding to CD28. Will
result in anergy and death.
Converted to TREG
Viruses (SARS-CoV-2)
Fungi (yeast)
Bacteria (intracellular)
Bacteria (extracellular)
Parasite (roundworm)
The immune system recognizes what has invaded the system, and generates the correct
response
Types of effector T cells
Extracellular
bacteria
In adaptive immunity, effector T cells work with other immune cells to
eliminate infections
B cells (antibodies); eosinophils, mast cells, and basophils; macrophages
Response to extracellular bacteria: Innate system, Antibodies, TH1, TH2, and TH17
cells
Pointy stick introduces bacteria. FIRST EXPOSURE
• Sentinal macrophages use TLR, etc to recognize pathogens and in response, secrete
inflammatory cytokines.
• The stab and the inflammatory cytokines increase vascular permeability. This releases
exudate which contains opsonins such as CRP, also complement, antimicrobial peptides.
• Complement can opsonize and lyse bacteria. Antimicrobial peptides can lyse cells.
• The inflammatory cytokines also cause extravasation of phagocytes (like neutrophils and more
macrophages) which eat phagocytose the bacteria and kill them using by lytic enzyme
digestion, reactive oxygen and nitrogen mechanisms, release of toxic granules, and NETosis.
• Complement products bind to mast cells and and macrophages causing release of more
inflammatory mediators, including prostaglandins and histamine, to enhance the response.
B cells in cortex (primary follicles) are
primed by Ag binding to surface Ig.
B cells go to border between cortex and
paracortex.and interact with the T cells.
Exudate carries Dendritic cells
and free antigen to lymph node
Dendritic cells activate TH cells.
TFH
TFH secrete IL-21 which helps primed B
cells proliferate and start class
switching, in combination with cytokines
from TH1 and TH2 cells.
TFH stay in the secondary
lymph tissue and reside in
the germinal centers with
memory B cells and FDC.
Help survival of memory
cells, and differentiation of
memory B cells into plasma
cells
TFH
TH1
Treg?
TH2
TH1,H2
Extracellular bacteria-what antibody effector
functions are needed?
Neutralization, opsonization, and complement
activation (IgG and IgM-TH1/TH2)
Will also need to recruit more phagocytes
from the blood to deal with the opsonized and
dead bacteria (IL-17-TH17)
The antibody response will
remove any remaining
bacteria, but also protect the
next time you get jabbed by
the pointy stick with the same
type of bacteria.
The antibodies will neutralize
the bacterial toxin (if any)
The antibodies will activate
complement-increasing
opsonization, and bacterial
lysis, as well as increasing
inflammation.
The antibodies will opsonize
the bacteria directly, increasing
phagocytosis
Embedded question: Name 3 types of TH effector cells used in the immune response against
extracellular bacteria
TH17 cells do not affect antibodies,
but are a part of the adaptive
response to extracellular bacteria.
Signal 3 for TH17 is IL-1, IL-6, TNF-a
(inflammatory cytokines)
TH17 contain the transcription factors
STAT3 and RORgT.
TH17 secrete IL-17 and IL-22.
IL-17 and IL-22 cause cells to secrete chemokines attract neutrophils,
Cause increased production on antimicrobial peptide (b-defensin)
Activate macrophages
Extravasation
This is basically a summary
of the entire course!
TH1 are also involved in more than helping B cells. When TH1 travel to the
site of the infection, they secrete inflammatory cytokines like TNF-a, and
also cytokines that activate macrophages, like IFN-g, so the macrophages
are more active phagocytes. The activated macrophages also can recruit
more neutrophils, and secrete more inflammatory substances.
The adaptive response to fungal
infections is similar to that for
intracellular bacteria. APC recognize
PAMPS and go to nodes to generate
TH1 and TH2 cells, which help B cells
develop into plasma cells that secrete
opsonizing, and some neutralizing
antibodies. TH1 cells travel to the site of
infection and activate macrophages to
increase phagocytosis and secrete
things that kill fungal cells.
TH17 cells are also generated in the
lymph nodes, and these also travel to
the site of infection and recruit
neutrophils and stimulate epithelial
cells to secrete antifungal peptides.
How does our immune system fight
intracellular bacteria?
• Our response starts out by the usual
route-an APC eats the bacteria by
phagocytosis. The macrophages and
DC detects the bacteria using PRR,
and start an innate response. A
dendritic cell eats the bacteria and
enough of it gets digested and
presented on MHC II to activate and
generate TH1 cells in the lymph node.
• At the site of infection, macrophages
will have eaten the bacteria, but
intracellular bacteria don’t allow much
fusion of the phagosome with the
lysosome, so the killing mechanisms
are not activated and the bacteria
aren’t killed. /A bit of digestion
happens, enough to put MHC II-Ag on
the cell surface.
The TH1 cells leave the lymph node
and end up in the blood. The activated
vascular endothelium at the site of
infection will allow the TH1 to
extravasate at the site of the infection,
where the infected macrophages are.
The infected macrophage bind the TH1
cell using TCR/MHC II-Ag, CD4/MHC
II, and this causes the TH1 cells to
secrete cytokines (IFN-g and TNF-b)
that will further activate the
macrophage, presumably upping the
reactive oxygen and nitrogen killing
mechanisms.
This is cell-mediated cytotoxicity, and it
could be enough to kill the intracellular
bacteria.
Or not….
The chemokines will recruit more
immune cells.
• In some cases, the bacteria is not killed
(such as M. tuberculosis, which is resistant
to the cell-mediated killing mechanisms of
the macrophage). However, the TH1 cell
and the now highly activated infected
macrophage send out signals to recruit
more TH1 cells and more macrophages.
These surround the original infected cell.
• The mass of activated macrophages and
TH1 cells are called a granuloma, or in the
case of tuberculosis, a tubercule. The
When the original
release of toxic substances from the
antigen/pathogen can’t be
infected macrophages and recruited
removed, this is called
neutrophils can cause a lot of tissue
Delayed-type, or type IV,
damage in the surrounding area.
hypersensitivity.
• TH1 also secrete cytokines that will cause
apoptosis, which is supposed to to kill the
If it is not a pathogen, it is
infected cell and release the trapped
called contact dermatitis.
bacteria, so that the surrounding
macrophages can try again to kill by
phagocytosis
• The granuloma also traps the bacteria so
they can’t escape and cause a systemic
infection.
• .
Poison ivy makes a compound that binds covalently to skin proteins.
When the proteins shed, they are ingested by APC, which then activate
TH1 cells. The TH1 cells go to the affected tissue, and activate
macrophages which try to get rid of the affected skin cells by releasing
inflammatory and other toxic compounds. Results in the red itchy rash.
Can be caused by metals as well as other things.
The rash can happen a week or so after exposure, but is more likely to
occur by activation of memory cells after the second exposure.
TFH?
TH1
IgA is the most prevalent Ig
produced in the gut, where
many TREG are.
Treg?
TH2
TH1,H2
We finally have antibody producing plasma cells! Some of the plasma cells are going to
circulate for a while, secreting antibodies to get rid of the infection, if the innate system hasn’t
already done it. Some will go to the bone marrow, where they live a long time, and produce
protective antibodies.
Plasma cells made in the MALT tend to stay there, and secrete IgA into the mucosa.
How do antibodies help get rid of infections? What type of infections???
Antibodies have 3 major effector
functions that help us fight disease:
1) Neutralization
2) Opsonization
3) Complement Activation
There are a few other functions that
we will hit in a few slides.
Neutralizing antibodies prevent the
antigen/pathogen from binding to a
cell’s surface. For example, an
antibody that bound to the SARSCoV-2 spike protein could
neutralize it, preventing it from
binding to the ACE2 receptor on
lung cells.
We have already learned about
opsonization and complement
activation (classical pathways was
activated by antibodies), and you
will see it represented in pictures
here.
More about neutralization:
In addition to binding to blocking
antigens from binding to a cell surface,
neutralizing antibodies can also bind
bacterial toxins and prevent them from
binding to cell receptors.
This is how the vaccine against diphtheria
and tetanus works. You get injected with
inactivated toxoid proteins and your body
builds antibodies that will neutralize the
toxin if you ever get infected with the
bacteria (Clostridium tetani,
Corynebacteria diphteriae).
Once the antigen is bound to neutralizing
antibodies, the stem (Fc) regions of the
antibodies can bind to Fc receptors on
macrophages, and the antigen is
destroyed in the lysosome. Opsonization
is similar, except a microbe is coated and
ingested, not just an antigen.
Neutralization also prevents toxins and pathogens from entering the body through
the mucosal layer. The Ig types that usually does this is IgA.
Neutralization 3 ways
Prevent a toxin from
binding to a receptor
Prevent a
virus from
binding to a
cell surface
receptor
Prevent a bacteria from binding to a cell’s surface
An effector function sort of related to opsonization is the ability of
antibodies to clear antigens complexes from blood or other places
in the body. This is related to complement, and is one of the
functions of complement we learned about earlier in the course.
Ab can bind soluble antigens, and form a larger complex where
the Ag cross-links multiple Ab together. In mucosal tissue, (lumen
of gut, for example) this will result in elimination.
In the blood, there is no obvious exit route for elimination.
However, the close spacing of the Ig Fc regions activates
complement via the classical pathway, and C3b is made. C3b lands
on the antigen/antibody complex.
The C3b can bind Complement Receptors (CRs)on red blood cells
or on phagocytes. The Fc portion of the Ab can bind Fc receptors
on macrophages in the spleen, which remove the complex and the
RBC.
Immune complexes can also cause problems (Type 2 and 3
hypersensitivity diseases).
So clearance of immune complexes is a antibody effector function.
Type III hypersensitivity-antibody complex disease
Immune complexes can be a problem-causing
inflammation. This is called Type III hypersensitivity.
It is one of the major pathologies found with some
auto-immune diseases. Example-Lupus,
Rheumatoid arthritis.
In Lupus, apoptosis leads to generation of
antibodies that react against self nuclear proteins
(this shouldn’t happen). Since we have a lot of self
proteins, there is a ton of antigen binding to the
auto-antibodies.
The antibodies make an immune complex, which
should get cleared.
But, if there is too much complex, the normal
methods of clearing them don’t work. The
complexes then bind to blood vessel walls, and
activate complement there, which causes
inflammation. They also can bind to the basement
membranes in the kidney glomerulus (filtering
tissue) and activate complement there too.
Inflammation. Results in kidney damage.
In RA, the immune complexes are deposited on
joint surfaces
•
When immune complexes are localized
by having antigens concentrated
intradermally, the type III
hypersensitivity reaction is the Arthus
reaction
•
It can happen with a booster shot of a
vaccine (when there are already
circulating antigens).
•
Mosquito bites are also an example of
the Arthus reaction
Other effector functions of antibodies:
• Antibody-dependent cell mediated cytotoxicity (ADCC). This involves antibodies
binding to non-self antigens on a host cell surface (like proteins produced as a
consequence of a virus infection, or cancer) or to a pathogen surface, such as a
helminth (worm). Effector cells (like NK cells, eosinophils) bind to the Fc portion
of the bound antibodies, and if this causes clustering of the Fc receptors, it starts a
signaling cascade that causes release of granules from the effector cell. Above, the
effector cell is an NK cell, which releases granzymes and perforins to kill the cell
by apoptosis, as we learned in the presentation about the innate response to viruses.
The effector cell could also be an eosinophil, which releases toxic and
inflammatory compounds.
• Mast cell sensitization. In this case,
an allergen or pathogen (again, this
happens with helminths) causes
production of antibodies (usually
IgE) that bind to Fc receptors on the
surface of mast cells. When the
sensitized mast cells encounter any
copies of the antigen in the body,
the bound immunoglobulins bind
the antigen, and this causes
clustering of the Fc receptors. This
sends a signal to the mast cell to
release granules containing
histamine and other inflammatory
substances.
This is the basis of the allergy
response.
The response can be local, or systemic.
If the response is systemic, (antigen gets into your blood) then you can get anaphylaxis
Engagement question: Name 5 effector functions of antibodies
IgE binds to mast cells
Each immunoglobulin class is suited for specific effector functions. The table
above summarizes which Ig are good at which functions. Remember, it is the Fc portion that
differs among the different Ig types.
Note that IgA is good at neutralization, not so good for ADCC. This is the Ig type made in
MALTs and secreted into the lumen of the intestines. Its job there is to prevent pathogens
from getting into the body through the GI tract.
IgGs (there are subclasses of IgG) are found in blood serum and are included in the exudate
that leaves the blood as part of the innate response to being jabbed with a pointy stick. They
are very good at opsonization and complement activation.
We will focus on activation of naïve TH cells for now.
Interaction between the TH cells and the APC will send
three signals to activate the naïve TH.
The first signal, called Signal 1, is when the TCR/CD3
complex recognizes and binds MHC II-Ag. CD4 binds
to MHC II. The signaling cascade is shown in a later
slide. This signaling cascade starts the activation
process.
Signal 2 is caused by CD28 on the T cell binding to
co-stimulator B7 on the APC. B7 is a dimer of B7.1
and B7.2. (B7.1 is also known as CD80. B7.2 is also
known as CD86). This activates more intracellular
signaling, and will lead to synthesis in the T cell of IL-2
and the IL2 receptor (among other things). IL-2
stimulates proliferation and survival.
Signal 3 is caused by the cytokines secreted by the
APC that tell the TH what type of effector cell it should
become.
Next we will go into each signal in more detail
•
Signal 2, the co-stimulatory signal, is
caused by CD28 on T cells binding to B7
on APC.
•
B7 binding to CD28 activates additional
kinases-including the MAP kinase
pathway. This results in activation of
additional transcription factors.
•
Instead NFAT causing cells to making
GRAIL, and become anergic, when Signal
1 combines with Signal 2, the two paths
converge to activate additional
transcription factors, including NF-kB
and AP-1, that work with NFAT to turn on
the genes for IL-2 and the IL-2 receptor
(IL-2R, also known as CD25).
•
If TCR-CD3-CD4/CD8 paths are activated
without the CD28-B7 interaction, the T
cells enter a state of ANERGY, which
means they can’t proliferate, and are
generally non-responsive.
•
Generation of anergic TH cells should
help prevent autoimmunity. B7 is only on
antigen presenting cells, and it is
increased by signaling from PRRs. If
there are no PAMPS, then. There is little
B7. No T cell activation.
Signal 2
B7
MHC-Ag
Signal 1
Nature Reviews Immunology 14, 435–446 (2014)
Signal three is the differentiation signal. It
is started when cytokines secreted by the
APC or other nearby cells bind to specific
cytokine receptors on to the T cell.
In many cases, the APC secretes specific
cytokines that work using the JAK-STAT
signaling pathway.
The STATs, which are specific for each
cytokine receptor, turn on genes encoding
key regulatory transcription factors (see
next slide), which then turn on the genes
specific to a type of TH effector cells, or
memory cell.
In many cases, the PRR on the APC that
were activated by the antigen/PAMPS
determine which cytokines are secreted for
Signal 3.
Signal 3
Cytokine for signal 3 from
APC and local
environment
Key regulatory
Transcription factor
Genes that are turned
on-the genes encode
these cytokines that are
secreted by the effector
TH cells.
The top row on the figure shows the cytokines that act as Signal 3. Note that some of the Signal 3
cytokines are inflammatory cytokines, which were made by the APC in response to TLR signaling.
Naïve TH cells differentiate into a type of effector TH cells (e.g. TREG, TFH,TH17, TH1, and TH2). We will
discuss these in later slides and also later in the course.
Each effector TH cells is characterized by a distinct regulatory transcription factor, that helps turn on
the genes needed for the cell to do its job. Among these gene are cytokines that are secreted by
the TH effector cells-each type has its own distinct set of cytokines that it secretes. We will discuss
these effector functions in detail a bit later in the course. Right now we need to talk more about
Signal 3.
Now we know how naïve TH (CD4+) cells are activated
and how they differentiate into the effector cells (TH1,
TH2, TH17,TFH, and TREG) (we didn’t discuss TH17 or TFH
much, because we do have time limits). We will talk
about what the effector cells do later in the course.
Virus-infected DC
The activation and differentiation happens in secondary
lymph tissue.
How are naïve TC (CD8+) cells activated, and how do
they differentiate into CTLs (cytotoxic T lymphocytes)?
Il-2R
Il-2
The simplest way is for a dendritic cell to become
infected by a virus at the site where the virus enters the
body (say, in the lungs, where someone inhaled
droplets containing SARS-CoV-2). We will come back
to how DC got this infection in a couple of slides.
The ribosomes in the infected DC will now start making
viral proteins. Some of the viral proteins will be
degraded by the Ub-proteasome system, and
presented on MHC I using the endogenous antigen
pathway.
The DC will go to the nearest lymph node (or other
secondary lymphoid tissue) and find Tc which, like TH,
are in the paracortex.
Once in the paracortex, the infected dendritic cell and
the Tc will test interactions (chemokines from DC
attract Tc)
Virus-infected DC
Signal 1 is MHC-1/Ag-TCR/CD3, and MHC I-CD8.
Signal 2 is B7-CD28.
The two signals combined activate NFAT, AP1, and
NFkB transcription factors to turn on the gene for IL-2
and IL-2 receptor. If you don’t have B7-CD28, only
NFAT is turned on, and you don’t get IL-2, you get
ANERGY. So far this is just like activation of TH cells.
Il-2R
IL-2 is secreted by the Tc cells, and it binds to the IL-2
receptor, causing the Tc to start proliferating.
Signal 3, which is not shown in the picture to the left, is
IL-12. IL-12 comes from the infected DC and is made
Il-2 when the viral PAMP activated a TLR . You can see
the pathway if you go to slide that has all the TLR
paths.
The activated Tc differentiates into a CTL. We will talk
about how CTLs work later in the course.
This is what one hopes will happen.
O’Neill LA, Bryant CE, Doyle SLTherapeutic targeting of toll-like receptors for infectious and inflammatory
diseases and cancer. Pharmacol Rev 61:177-197
This is to remind you of the TLR pathways. IL-12 is another inflammatory cytokine that is made when
PAMPS bind to the TLRs.
Sometimes, the Tc can’t be activated without help. It
depends on what the virus is, apparently. The problem
seems to be that some viruses do not stimulate B7
production enough in the DC, -so signal two is too low.
In this case, the Tc needs help (cytokines) from a
nearby TH CD4 cell. This is usually a TH1 cell.
The CD4 cell is activated by the same DC as the Tc.
This can happen because:
1) The DC can phagocytose the virus as well
as be infected by the virus. This would allow
the viral proteins to be processed by the
exogenous pathway and presented on MHC
II.
2) The crossover pathway of Ag
presentation, which I told you to not worry
about. You can go back and look at it if you
want.
TH cells will bind to the same APC as the Tc (but will
bind to MHC II-Ag). They will bind whatever B7 is
there. It takes less signal 2 to activate TH cells than Tc
cells (or they coud already be TH1 cells and not need
much activation). Signal 2, in addition to turning on the
IL-2 gene, will turn on expression of another signaling
protein called CD40L, which binds to CD40 on the APC.
Binding of CD40L to CD40 starts a signaling cascade
that results in the APC making more B7. It also causes
the APC to make 4-IBBL, which is a member of the
TNF family and is another co-stimulator of Tc cells. It
binds to 4-IBB on the Tc.
The extra co-stimulation will cause the Tc to also turn
on the genes for IL-2 and IL-2R. This, combined with
the extra IL-2 from the TH cell, drives Tc proliferation
and survival.
IL-12 from the APC will still be signal 3 to differentiate
into CTL. It could also be signal 3 for the TH cells and
cause them to become TH1 cells, if they are not already
differentiated.
DC-SIGN
We have talked briefly about how viruses infect only certain cells because the bind to surface
protein receptors that are only expressed on certain cell types. For example, SAS-CoV-2
infects cells that have the ACE2 receptor on their surface.
So how can Dendritic cells be infected with viruses to present on MHC I, if they don’t have the
specific viral receptor??
It turns out that DC have sticky proteins on their surface, called DC-SIGNs. Most viruses can
bind to these and use this receptor to infect the DC. This allows DC to present most viral
antigens on MHC I, so that CTL that are specific to that virus can form.
The virus shown in this picture is HIV, and it also binds to other things. Don’t worry about that
right now.
Types of effector T cells
We have finally made T cells that can DO SOMETHING!! We’ll talk about what they
do to keep us healthy right after we activate some B cells.
•Effector TH (usually TH1 or TH2) cells are now
in the secondary lymph tissue-ready to help
the B cells.
•Now it’s time to activate B cells and have
them differentiate into a plasma cells that
secrete the correct type of antibody, and
memory cells
•The process is called antigen-dependent B
cell maturation
•The Antigen-independent phase of B-cell
maturation is the development of the naïve B
cell in the bone marrow, and we’ll discuss
that later in the course
Three signal...
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