Basic Immunology
Innate Immunity
Granulocytes, Monocytes/Macrophages, DCs, ILCs, complement. Quick (6-24 hours) but not specific response.
Innate immunity depends on macrophages or DCs, which do not possess rearranged receptors and have limited
repertoire receptors. They’re activated by alarmins and PAMPs/DAMPs. The principal receptors that recognize
products of microbial pathogens (pattern recognition receptors-PRRs) are Toll-like receptors (TLRs), the NOD–like
family receptors (NLRs), the RIG-I-like family receptors (RLRs) and C-type lectin receptors (CLRs). Signaling sensors
are expressed on the cell surface, in the endosome, or in the cytosol, where they recognize their corresponding
ligands → NF-kB, MAPKs etc. → expression of cytokines or co-stimulatory molecules (CD40, CD86). Note that PRRs
are expressed also on lymphocytes (B, T, memory).
Alarmins
Alarmins are DAMPs constitutively available and passively released from stressed, injured or dying cells upon
infection or tissue injury. They alert the immune system even in the absence of infection. Examples: heat-shock
proteins, high-mobility group protein B1 (HMGPB1), calprotectin, IL-1α and antimicrobial peptides like defensins .
PAMPs
Molecules found on microbes, not normally present in the host: LPS (from Gram[-] bacterial cell walls),
peptidoglycans and lipoteichoic acid (from Gram[+] bacteria), flagellin (from bacterial flagella protein), bacterial
unmethylated CpG DNA, dsRNA (from viruses), mannans & β-glucans (from fungal cell walls).
DAMPs
Nucleic acids, lipids such as saturated fatty acids or oxidized LDL, proteins such as heat shock proteins or S100
family proteins, K+ efflux, ATP and MSU crystals, cholesterol and asbestos
Receptor types
3 categories of sensors: signaling (TLRs and cytosolic [RLRs, NLRs]), internalizing (CLRs), soluble (opsonins,
complement, MBL).
1. SIGNALING
Toll-like receptors
Constitutively expressed in innate immunity cells (DCs, macrophages, neutrophils) as well as epithelial cells and B-
cell but can also inducibly be expressed on other cells (ex. fibroblasts). The type of TLR expressed depends on the
cell and it’s activation status.
Lipid and protein-recognizing TLRs are located in the cell membrane, while nucleic acid–recognizing ones
are in the ER and upon activation are transported to the endosome, where nucleic acids are released from virus
or virally infected cells. They play major roles in activating APCs like DCs and B-cells to express costimulatory
molecules. Extracellular domain contains LRR, intracellular region contains a TIR domain (a common structure in
TLR and IL-1R family members). TLRs have no enzymatic activity, but bind adapters like MyD88 and TRIF. TLR
ligands are lipids, proteins and nucleic acids.
Lipids
-TLR1/2/6: lipopeptides (Gram (+) bacteria, fungi, mycoplasma)
-TLR4: LPS, found in the outer cell walls of Gram (-) bacteria. LPS is first bound to LPS-binding protein (a
soluble factor) in the serum and transferred to target cells such as macrophages. Macrophages express CD14
which can capture and retain LPS. LPS then activates TLR4.
in obesity, adipocytes secrete chemotactic factors that recruit macrophages and also produce
saturated fatty acids that activate recruited macrophages via TLR4 to produce TNF which then further activates
,adipocytes to produce saturated fatty acids. Thus obesity is associated with a low-grade chronic inflammation
caused by the loop between adipocytes and macrophages.
Proteins (TLR5): ex. flaggelin
Nucleic acids (TLR3/7/8/9) (in endosomes):
-TLR3: dsRNA (in RNA-virus-infected cells)
-TLR7/8: ssRNA (influenza, HIV)
-TLR9: unmethylated CpG DNA (unmethylated CpG motif is more abundant in bacterial DNA than in
mammalian DNA; mammalian CpG DNA is often methylated). Therefore unmethylated CpG DNA can be regarded
as non-self and is recognized by TLR9. The self unmethylated CpG DNA that is recognized by TLR9 doesn’t cause
immunostimulation because it’s degraded before reaching the endosomes and fails to reach endosomes.
Cytosolic sensors
RLRs = (RIG-I)–like receptors: RIG-I, MDA5 and other. They are potent IFN-I inducing sensors. RIG-I and
MDA-5 both carry CARD domains and can recognize virally derived RNAs. MDA5 is critical for sensing
picornaviruses.
NLRs = (NOD)-like receptors: NLR family is composed of 22 members that respond to diverse products,
including bacterial/ viral products and DAMPs (K+ efflux, ATP, MSU crystals, cholesterol, asbestos). They carry a
CARD, a PYD domain or a BIR domain as a protein interaction domain at the N-terminal, which is followed by an
NACHT nucleotide-binding domain and an LRR domain. LRRs are believed to function in ligand sensing and
autoregulation, whereas CARD and PYD domains mediate homotypic protein-protein interactions for downstream
signaling. The NACHT domain, which is the only domain common to all NLR family members, enables activation of
the signaling complex via ATP-dependent oligomerization.
PYHIN= (PYN) and (HIN)–containing protein: ex. AIM2 and IFI16, detect dsDNA.
cGAS (=cGAMP synthase [=cyclic GMP-AMP]): a cytoplasmic enzyme that senses dsDNA and generates
GAMP (from ATP/GTP) and leads to expression of IFN-I, via STING.
Some PAMPs are recognized by both transmembrane and cytosolic signaling sensors.
2. INTERNALIZING SENSORS
are expressed on the surface of neutrophils, macrophages or DCs helping phagocytosis or uptake of ligands for
further processing. They bind to and internalize PAMPs or microorganisms, which are subsequently transported
to lysosomal compartments by phagocytosis where organism-derived proteins are degraded or processed for
presentation to T-cells.
CLRs =C-type lectin receptors: lectins are proteins recognizing carbohydrates (ex. mannose, β-glucans)
and some lectins function as internalizing sensors. The mannose receptor is a transmembrane protein (on
macrophages and DCs) that binds terminal mannose and fructose residues on microbial cell walls, internalizes
them and facilitates phagocytic clearance, cytokine release and activation of immune cells.
Scavenger Receptors
Complement Receptors
Fc Receptors (FcRs)
(Some TLRs [ex. TLR2/4] can also promote endocytosis after ligand binding, even if uptake isn’t their
primary role).
3. SOLUBLE SENSORS
Pentraxins (CRP, serum amyloid P): act as opsonins, produced by macrophages or hepatocytes and bind
to the cell wall of microorganisms, thus designating them as targets for phagocytosis. Soluble sensors can fix C1q
and activate the complement to opsonize a pathogen, without the need of an antibody. FcγRI and FcγRIIa also
bind CRP and serve as its receptors, opsonizing without the need of an antibody.
CRP: elevation occurs within 4h of tissue injury and peaks within 24–72h, with a T1/2 =18h (in
contrast, ESR falls within 1 week).
, Mannose-binding lectin (MBL): a soluble molecule that binds mannose residues on pathogens and
triggers lectin-pathway of complement cascade.
Complement: 3 pathways: classic, alternative, lectin, all lead to cleavage of C3 into C3a + C3b. C3b
opsonizes bacteria, that are further phagocytosed or attacked by MAC.
Activation of adaptive immunity on infection
Locally activated APCs mature to express a distinct set of chemokine receptors; then they are transported to the
lymph nodes, where they interact with and activate T-cells. Clonal T-cell expansion requires not only
TCR–mediated signaling but also costimulatory molecule–mediated. Internalizing sensors facilitate antigen
presentation and signaling sensors such as TLRs can enhance expression of costimulatory molecules, including
CD80/CD86. APCs are critically involved in regulating Th differentiation. This activity depends on tissue origin, cell
subsets or maturation stage of the APCs, but the nature of the stimuli activating the APCs is most important.
Antiviral responses
Response to viral infections by production of IFNs can also upregulate expression of the MHC and induce DC
maturation. Cytosolic sensors such as RLRs are potent IFN-I-inducing sensors. Among TLRs, TLR7 and TLR9 can
induce pDCs to produce IFN-I.
-pDCs are a DC subset known as IFN-α–producing cells, look like plasma cells and exhibit poor antigen-
presenting activity owing to low levels of expression of MHC-II and costimulatory molecules. They express TLR7/9
and are involved in antiviral immunity by secreting IFN-I (esp. IFN-α) in a TLR7/9-dependent manner. Because
they are capable of activating other immune cells, pDCs serve as a bridge between innate and adaptive immunity.
A pDC's ability to stimulate T cells is heightened following maturation which includes expression of both MHC-I
and - II molecules.
-Cross-presentation: exogenous antigen presentation through MHC-I to generate CD8+ T-cell responses.
Virally infected cells are ingested by APCs and antigens are associated with MHC-I and –II. The cross-presentation
system ensures that virally infected cells, which do not always possess the ability to present antigen by
themselves, can be targets for cytotoxicity. Also, for cancer surveillance, because most cancer cells show poor
antigen presentation ability (downregulation of MHC-I).
-NK response: NK cells kill cells with reduced MHC-I and produce IFN-γ enhancing antiviral immunity.
Molecular mechanism of sensor signaling
TLRs carry the intracytoplasmic TIR region that associates with cytoplasmic adapters that also possess
the TIR region (MyD88, TIRAP, MAL, TRIF, TRAM etc), leading to activation of NF-kB or IRFs, via pathways that
include kinases like IKK, IRAK or an adapter molecule (TRAF6), causing proinflammatory cytokines and IFN-I gene
expression. IKK family members play critical roles in linking TLR signaling and activation of transcription factors.
-MyD88 associates with all TLRs except TLR3 and cooperates with TIRAP downstream of TLR2/4. It
is critical for all TLR7/9-induced effects, including IFN-I production. Steps: MyD88 recruitment → IRAKs
recruitment and activation of TAK1 → phosphorylation of IKK (IκB kinase complex) → release of NF-kB and
translocation to the nucleus for gene expression induction.
-TRIF pathway (TLR3/4): recruitment of TRIF, activation of TRAF3, phosphorylation of TBK1 and
IKKε, activation of IRF3 which translocates to the nucleus and induces IFN-I production. TRAM is required for TLR4
to associate with TRIF. TLR3 can directly associate with TRIF and does not require TRAM for its signaling.
RLRs do not use adapters with the TIR region but require adapters carrying a CARD-like domain (ex. IPS-1),
to induce cytokine and IFN expression, via NF-kB and IRF.
cGAS generates cGAMP, which activates the ER adaptor protein STING (→IFN).
Inflammasome
The inflammasomes are large intracellular protein complexes that aim to activate the pro-caspase to caspase and
convert inactive pro-IL 1β into its highly inflammatory mature form IL-1β. Other proteins cleaved by caspase are
Innate Immunity
Granulocytes, Monocytes/Macrophages, DCs, ILCs, complement. Quick (6-24 hours) but not specific response.
Innate immunity depends on macrophages or DCs, which do not possess rearranged receptors and have limited
repertoire receptors. They’re activated by alarmins and PAMPs/DAMPs. The principal receptors that recognize
products of microbial pathogens (pattern recognition receptors-PRRs) are Toll-like receptors (TLRs), the NOD–like
family receptors (NLRs), the RIG-I-like family receptors (RLRs) and C-type lectin receptors (CLRs). Signaling sensors
are expressed on the cell surface, in the endosome, or in the cytosol, where they recognize their corresponding
ligands → NF-kB, MAPKs etc. → expression of cytokines or co-stimulatory molecules (CD40, CD86). Note that PRRs
are expressed also on lymphocytes (B, T, memory).
Alarmins
Alarmins are DAMPs constitutively available and passively released from stressed, injured or dying cells upon
infection or tissue injury. They alert the immune system even in the absence of infection. Examples: heat-shock
proteins, high-mobility group protein B1 (HMGPB1), calprotectin, IL-1α and antimicrobial peptides like defensins .
PAMPs
Molecules found on microbes, not normally present in the host: LPS (from Gram[-] bacterial cell walls),
peptidoglycans and lipoteichoic acid (from Gram[+] bacteria), flagellin (from bacterial flagella protein), bacterial
unmethylated CpG DNA, dsRNA (from viruses), mannans & β-glucans (from fungal cell walls).
DAMPs
Nucleic acids, lipids such as saturated fatty acids or oxidized LDL, proteins such as heat shock proteins or S100
family proteins, K+ efflux, ATP and MSU crystals, cholesterol and asbestos
Receptor types
3 categories of sensors: signaling (TLRs and cytosolic [RLRs, NLRs]), internalizing (CLRs), soluble (opsonins,
complement, MBL).
1. SIGNALING
Toll-like receptors
Constitutively expressed in innate immunity cells (DCs, macrophages, neutrophils) as well as epithelial cells and B-
cell but can also inducibly be expressed on other cells (ex. fibroblasts). The type of TLR expressed depends on the
cell and it’s activation status.
Lipid and protein-recognizing TLRs are located in the cell membrane, while nucleic acid–recognizing ones
are in the ER and upon activation are transported to the endosome, where nucleic acids are released from virus
or virally infected cells. They play major roles in activating APCs like DCs and B-cells to express costimulatory
molecules. Extracellular domain contains LRR, intracellular region contains a TIR domain (a common structure in
TLR and IL-1R family members). TLRs have no enzymatic activity, but bind adapters like MyD88 and TRIF. TLR
ligands are lipids, proteins and nucleic acids.
Lipids
-TLR1/2/6: lipopeptides (Gram (+) bacteria, fungi, mycoplasma)
-TLR4: LPS, found in the outer cell walls of Gram (-) bacteria. LPS is first bound to LPS-binding protein (a
soluble factor) in the serum and transferred to target cells such as macrophages. Macrophages express CD14
which can capture and retain LPS. LPS then activates TLR4.
in obesity, adipocytes secrete chemotactic factors that recruit macrophages and also produce
saturated fatty acids that activate recruited macrophages via TLR4 to produce TNF which then further activates
,adipocytes to produce saturated fatty acids. Thus obesity is associated with a low-grade chronic inflammation
caused by the loop between adipocytes and macrophages.
Proteins (TLR5): ex. flaggelin
Nucleic acids (TLR3/7/8/9) (in endosomes):
-TLR3: dsRNA (in RNA-virus-infected cells)
-TLR7/8: ssRNA (influenza, HIV)
-TLR9: unmethylated CpG DNA (unmethylated CpG motif is more abundant in bacterial DNA than in
mammalian DNA; mammalian CpG DNA is often methylated). Therefore unmethylated CpG DNA can be regarded
as non-self and is recognized by TLR9. The self unmethylated CpG DNA that is recognized by TLR9 doesn’t cause
immunostimulation because it’s degraded before reaching the endosomes and fails to reach endosomes.
Cytosolic sensors
RLRs = (RIG-I)–like receptors: RIG-I, MDA5 and other. They are potent IFN-I inducing sensors. RIG-I and
MDA-5 both carry CARD domains and can recognize virally derived RNAs. MDA5 is critical for sensing
picornaviruses.
NLRs = (NOD)-like receptors: NLR family is composed of 22 members that respond to diverse products,
including bacterial/ viral products and DAMPs (K+ efflux, ATP, MSU crystals, cholesterol, asbestos). They carry a
CARD, a PYD domain or a BIR domain as a protein interaction domain at the N-terminal, which is followed by an
NACHT nucleotide-binding domain and an LRR domain. LRRs are believed to function in ligand sensing and
autoregulation, whereas CARD and PYD domains mediate homotypic protein-protein interactions for downstream
signaling. The NACHT domain, which is the only domain common to all NLR family members, enables activation of
the signaling complex via ATP-dependent oligomerization.
PYHIN= (PYN) and (HIN)–containing protein: ex. AIM2 and IFI16, detect dsDNA.
cGAS (=cGAMP synthase [=cyclic GMP-AMP]): a cytoplasmic enzyme that senses dsDNA and generates
GAMP (from ATP/GTP) and leads to expression of IFN-I, via STING.
Some PAMPs are recognized by both transmembrane and cytosolic signaling sensors.
2. INTERNALIZING SENSORS
are expressed on the surface of neutrophils, macrophages or DCs helping phagocytosis or uptake of ligands for
further processing. They bind to and internalize PAMPs or microorganisms, which are subsequently transported
to lysosomal compartments by phagocytosis where organism-derived proteins are degraded or processed for
presentation to T-cells.
CLRs =C-type lectin receptors: lectins are proteins recognizing carbohydrates (ex. mannose, β-glucans)
and some lectins function as internalizing sensors. The mannose receptor is a transmembrane protein (on
macrophages and DCs) that binds terminal mannose and fructose residues on microbial cell walls, internalizes
them and facilitates phagocytic clearance, cytokine release and activation of immune cells.
Scavenger Receptors
Complement Receptors
Fc Receptors (FcRs)
(Some TLRs [ex. TLR2/4] can also promote endocytosis after ligand binding, even if uptake isn’t their
primary role).
3. SOLUBLE SENSORS
Pentraxins (CRP, serum amyloid P): act as opsonins, produced by macrophages or hepatocytes and bind
to the cell wall of microorganisms, thus designating them as targets for phagocytosis. Soluble sensors can fix C1q
and activate the complement to opsonize a pathogen, without the need of an antibody. FcγRI and FcγRIIa also
bind CRP and serve as its receptors, opsonizing without the need of an antibody.
CRP: elevation occurs within 4h of tissue injury and peaks within 24–72h, with a T1/2 =18h (in
contrast, ESR falls within 1 week).
, Mannose-binding lectin (MBL): a soluble molecule that binds mannose residues on pathogens and
triggers lectin-pathway of complement cascade.
Complement: 3 pathways: classic, alternative, lectin, all lead to cleavage of C3 into C3a + C3b. C3b
opsonizes bacteria, that are further phagocytosed or attacked by MAC.
Activation of adaptive immunity on infection
Locally activated APCs mature to express a distinct set of chemokine receptors; then they are transported to the
lymph nodes, where they interact with and activate T-cells. Clonal T-cell expansion requires not only
TCR–mediated signaling but also costimulatory molecule–mediated. Internalizing sensors facilitate antigen
presentation and signaling sensors such as TLRs can enhance expression of costimulatory molecules, including
CD80/CD86. APCs are critically involved in regulating Th differentiation. This activity depends on tissue origin, cell
subsets or maturation stage of the APCs, but the nature of the stimuli activating the APCs is most important.
Antiviral responses
Response to viral infections by production of IFNs can also upregulate expression of the MHC and induce DC
maturation. Cytosolic sensors such as RLRs are potent IFN-I-inducing sensors. Among TLRs, TLR7 and TLR9 can
induce pDCs to produce IFN-I.
-pDCs are a DC subset known as IFN-α–producing cells, look like plasma cells and exhibit poor antigen-
presenting activity owing to low levels of expression of MHC-II and costimulatory molecules. They express TLR7/9
and are involved in antiviral immunity by secreting IFN-I (esp. IFN-α) in a TLR7/9-dependent manner. Because
they are capable of activating other immune cells, pDCs serve as a bridge between innate and adaptive immunity.
A pDC's ability to stimulate T cells is heightened following maturation which includes expression of both MHC-I
and - II molecules.
-Cross-presentation: exogenous antigen presentation through MHC-I to generate CD8+ T-cell responses.
Virally infected cells are ingested by APCs and antigens are associated with MHC-I and –II. The cross-presentation
system ensures that virally infected cells, which do not always possess the ability to present antigen by
themselves, can be targets for cytotoxicity. Also, for cancer surveillance, because most cancer cells show poor
antigen presentation ability (downregulation of MHC-I).
-NK response: NK cells kill cells with reduced MHC-I and produce IFN-γ enhancing antiviral immunity.
Molecular mechanism of sensor signaling
TLRs carry the intracytoplasmic TIR region that associates with cytoplasmic adapters that also possess
the TIR region (MyD88, TIRAP, MAL, TRIF, TRAM etc), leading to activation of NF-kB or IRFs, via pathways that
include kinases like IKK, IRAK or an adapter molecule (TRAF6), causing proinflammatory cytokines and IFN-I gene
expression. IKK family members play critical roles in linking TLR signaling and activation of transcription factors.
-MyD88 associates with all TLRs except TLR3 and cooperates with TIRAP downstream of TLR2/4. It
is critical for all TLR7/9-induced effects, including IFN-I production. Steps: MyD88 recruitment → IRAKs
recruitment and activation of TAK1 → phosphorylation of IKK (IκB kinase complex) → release of NF-kB and
translocation to the nucleus for gene expression induction.
-TRIF pathway (TLR3/4): recruitment of TRIF, activation of TRAF3, phosphorylation of TBK1 and
IKKε, activation of IRF3 which translocates to the nucleus and induces IFN-I production. TRAM is required for TLR4
to associate with TRIF. TLR3 can directly associate with TRIF and does not require TRAM for its signaling.
RLRs do not use adapters with the TIR region but require adapters carrying a CARD-like domain (ex. IPS-1),
to induce cytokine and IFN expression, via NF-kB and IRF.
cGAS generates cGAMP, which activates the ER adaptor protein STING (→IFN).
Inflammasome
The inflammasomes are large intracellular protein complexes that aim to activate the pro-caspase to caspase and
convert inactive pro-IL 1β into its highly inflammatory mature form IL-1β. Other proteins cleaved by caspase are
