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Cytokines and Thelper subsets
MCB 150, Pr Coscoy
I. Characteristics of Cytokines
(CKs)
CKs are small proteins (<30 kDa)
CKs are similar to hormones and growth
factors.
CKs control the immune system.
 Cytokines include subsets called lymphokines, monokines,
chemokines, etc.
Cytokine Network
CKs are part of complex system that
regulates the immune system.
CKs are primarily produced by immune
system but many other organs (liver, brain,
endocrine glands) make CKs to influence
immune response.
Cytokine Network
T helper and
Macrophage
Interaction
highlighted
Effector T cell
Th cells control immune
system through CKs.
Activation of Cells
= Production of Cytokines
Production of CKs is usually part of immune cell
activation.
Cytokines are produced in response to different
stimuli (e.g antigen receptor, cytokine/chemokine
receptor).
Cytokine Network on a Local Level
IL-4 Re
IL-4
T cell
B cell
IL-2 gene
IL-4 gene
IL-2 Receptor
IL-2
II. Measuring Cytokines
Protein amount by ELISA.
Good for in vitro experiments.
Protein amount by bioactivity assay using
CK dependent cell lines.
RNA message by PCR.
Antigen Capture ELISA for IL-2
III. Actions of Cytokines
Autocrine acts on same cell that produced it.
IL-2 for T cell activation
Paracrine acts on nearby cells.
T cell help for B cells
Endocrine acts on cell at a distance (through
bloodstream). Inflammatory cytokines.
Actions of Cytokines
IL-2 for
T-cell activation
T-cell help
for B cells
Inflammatory cytokines
IV. Properties of Cytokines
Pleiotropy
Same cytokine has different effects on cells
can be activating or inhibiting.
Example: IL-10 can be inhibitory to
macrophages and Th1 cells yet activating
for Th2 cells and B cells.
Synergy
Experimental Example
Proliferation of CTLLs
(CTLLs are cytokine dependent cell line)
Both IL-2 or IL-4 activate CTLLs in vitro.
Maximal proliferation obtained when both
IL-2 and IL-4 added.
Both cytokines need to be blocked in order
to inhibit growth.
Properties of
Cytokines
Pleiotropy
Redundancy
Synergy
Antagonism
CK
IL-1
Secreted
Primary target
Hallmark
IL-2
Macs,
endothelium
Th cells
Increases CAMs
Activates macs
Th1, Tc, NK cells
ProInflammatory
Th1 cy tokine
IL-4
Th2 cells
Th2 cells, B cells
Th2 cy tokine
IL-6
Macs Th2 ce lls Th2 cells, B cells
ProInflam/Th2
IL-7
Bone marrow
Stem cells
Hematopoiesis
IL-8
Neutrophils
Endothelial cells
Chemokine
IL-10
Th2 cells,
Macs, Th2 and B cells Th2 cy tokine
IL-12
Macrophages,
dendritic cells
TNF- Macs, T, B
endothelial
TNF- Th1 cells
IFN
IFN 
Neutrophils,
endo/epithelial
Th1 cells,
macrophages
Th1, Tc, NK cells
Th1 cy tokine
tumor cells, macs,
B cells, T cells
macrophages, Th1
cells,
Uninfected cells
ProInflammatory
Inflammatory
macrophages, Th1
Anti-viral
cytokines
Th1 cy tokine
Inflammatory
V. Functions
Of Cytokines
VI. CD4+ T helper Subsets
Th1/Th2 Cytokine Bias
CD4+ Thelper cells can be divided into subsets
based on their cytokine production.
Th1 cells produce IL-2, IFN-, TNF-
CKs which activate cell mediated immunity
Th2 cells activate IL-4, IL-6, IL-10
CKs that activate humoral immunity
These Th subsets were originally identified using mouse T cell clones.
Mouse Th Subset
Cytokine
Th1 Th2
Table 12-4
from Goldsby
Th0 ---> Th1 or Th2
Original mouse experiments on Th cells
(Mosmann et al (DNAX) 1986 J Immunol)
Antigen specific T cells placed in culture with
antigen and APCs to make T cell lines.
Spleen cells (Th0) add IL-12  Th1 cells
IL-2, IFN-, TNF-
Spleen cells (Th0) add IL-4  Th2 cells 
IL-4, IL-6,IL-10
 (Th0 --precursor cell that produces IL-2, IL-4, and IFN-.)
Th1/Th2
Naïve Th0
IL-12
Effector
Th1 cell
IL-2, IFN-
IL-2, IL-4, IFN-
IL-4
Effector
Th2 cell
IL-4, IL-6, IL-10
Th1/Th2 Antagonism
IL-4 blocks Th1
IL-4
IL-12
Th1 cell
IL-2, IFN-
IFN- blocks Th2
IFN-
IL-4
Th2 cell
IL-4, IL-6, IL-10
Th1/Th2 Regulation
T-bet is a transcription factor that is required
for Th1 specific genes such as IL-12R
IL-4 blocks T-bet
IL-12
Th1 cell
IL-2, IFN-
IFN-
Enhances T-bet
Th2 cell
IL-4, IL-6, IL-10
IL-4 vs IFN-
T-bet (Th1 associated)
activated by IFN- and
turned off by IL-4.
Conversely in Th2
transcription factor
GATA-3 activated by IL4 turned off by IFN-.
Role for Th1 vs Th2 in
Immune Response
Both subsets activated in lymph nodes (LN)
immune responses to complex antigens.
Th1 cells leave LN to find activated
endothelium tissue to activate macrophages.
Th2 cells can stay in LN to activate B cells.
What controls Th1 vs Th2?
1) Amount of antigen.
Mouse experiments originally showed high dose for
Th1.
2) MHC and TCR affinity.
High affinity TCR = Th1.
3) Dendritic
cell subsets during activation.
APC subsets activate Th1 or Th2 preferentially.
4) Toll-like receptor activation.
Influence of APC Subsets on Th1/ Th2
Dendritic cell
Myeloid-like dendritic cells produce
abundant IL-12 and drive Th1.
Lymphoid-like dendritic cells produce low
levels of IL-12 are permissive for Th2.
Toll-like receptors (TLRs)
Influence of APCs on Th1/ Th2
Evidence for TLR activation influencing
Dendritic cell maturation.
– TLR9 binds bacterial CpG DNA
– TLR4 binding to bacterial heat shock proteins
TLR activation induces APC expression of
IL-12, IL-23, IL-27
Th1
TLR vs IL-12
in Th1/ Th2 development
New evidence suggests that TLR activation
influencing Th1 outcome through initiation
of TLR adapter molecule MyD88.
May be more important than IL-12 for Th1.
MyD88-/- mice fail to control
acute Toxoplasma infection
Survival curve shows MyD88 is just as important as IL-12 for
Th1 response after Toxoplasma infection.
Control mice ( )
MyD88-/- mice ( )
IL-12p40-/- mice ( )
Scanga et al The Journal of Immunology,
2002, 168: 5997-6001.
MyD88-/- mice default to Th2
Role for TLR Activation in Th1/Th2
IFN-
A. MyD88-/- mice
Response to
Th2 pathogen
B. MyD88-/- mice
Response to
Th1 pathogen
C. WT mice
Response to
Th1 pathogen
IL-4
IL-5
IL-10
IL-13
Th Cytokine Bias in Disease
Examples
Leishmania in mice (Richard Locksley)
 C57Bl.6 mice mice have Th1 immune response and
resolve infection.
 BALB/c mice produce Th2 cytokines unable to control
Leishmania lesions.
Leprosy in Humans (Robert Modlin)
 Tuberculoid form has Th1 response and limits disease
(healing).
 Lepromatous form has Th2 response and uncontrolled
disease (leprosy).
Th Cytokine Bias in Disease:Leprosy
Skin disease caused by Mycobacterium leprae
Tuberculoid: has Th1
response and limits disease
(healing).
Lepromatous: has Th2
response and uncontrolled
disease (leprosy).
Cytokine Bias in Leprosy
RNA from skin lesions of patients
Cytokines as Ligands
T
Cell
Ligand
APC
Target
Receptor
TNF- or Lymphotoxin
Soluble
or membrane bound
LT  and  Receptors
VII. Cytokine Receptors
Expression of cytokine receptors controls
the ability of a cytokine to act on a cell.
Cell activation increases cytokine receptor
expression.
Cytokine Receptor
Families
5 different families of receptors
based on common structural motifs.
--> see book for more details
IL-2 Receptor Subfamily
Shared common  subunit
Only IL-2 and IL-15 have unique alpha subunit
X-Linked SCID
Common  chain Deficiency
Mutation in  chain so unable to signal
through IL-2, IL-4, IL-7, IL-9, IL-15.
No T cells abnormal thymus.
Immunocompromised
susceptible to infections.
SCID Patient with severe Candida in mouth.
GM-CSF Receptor Subfamily
Hematopoietin Receptors
Low affinity
receptors
compete for
common  subunit
to get high affinity
binding.
GM-CSF
Granulocyte monocyte
Colony stimulating factor
GM-CSF Receptor Subfamily
Hematopoietin Receptors
IL-3, IL-5
GM-CSF
activate
common
 subunit.
Cytokine Receptor Signal through JAKs and STATs
Cytokine Receptor Signaling
JAKs and STATs (Model of Signal)
Binding of cytokine ligand
brings together receptor subunits
JAKs (Janus Associated Kinases)
are tyrosine kinases that
phosphorylate tyrosines.
STAT (Signal Transducers and
Activators of Transcription )
dimerize for function.
JAKs and STATs
Usage by CK Receptors
Overlapping and Unique
JAK1 is commonly used
by CKs from completely
different CKR families
STAT6 is
ONLY used
IL-4.
The Yin and Yang
of Th1 and Th2
Immune Responses