Interferon-gamma and immune system

By
Wat Mitthamsiri, MD.
Allergy and Clinical Immunology Fellow
King Chulalongkorn Memorial Hospital
Interferon-Gamma
And
Immune System

Outline
• Introduction
• What is interferon (IFN)
• Classification of IFN
• Interferon gamma (IFN-γ)
– History and biology
– Roles with other immune components
– Roles in infection defense
– Roles in autoimmunity
– Roles in allergy and hypersensitivity

What is interferon (IFN)
• Interferons are proteins which produce
antiviral and antiproliferative responses in
cells.
• On the basis of their sequence interferons
are classified into five groups: α, α-II (or
omega), β, delta (or trophoblast) and γ.
• Except for γ-interferon, the sequence of
all the others are related
PROSITE documentation PDOC00225, Swiss Institute of Bioinformatics, http://prosite.expasy.org/cgi-bin/prosite/prosite-search-ac?PDOC00225#ref1
InterPro: protein sequence analysis & classification, EMBL-EBI, http://www.ebi.ac.uk/interpro/entry/IPR000471

What is interferon (IFN)
• Roles of IFN:
– Decrease tumor growth, inflammation, and
angiogenesis
– Innate immunity (IFN-α and IFN-β)
– Adaptive immunity (IFN-γ)
O Meyer, Joint Bone Spine 76 (2009) 464–473

Classification of IFN
• 3 main classes:
K M Pollard, et al., Discov Med, 2013, 16(87):123-131.

• Type I IFNs
– Encoded by 17 nonallelic genes
• Lack introns
• Located on chromosome 9 in humans
– Glycosylated proteins,160-200 amino acids
– Sharing 30% to 55% homology
• Type II IFN
– 140 amino acids and shares no homology
with type I IFNs

• Type III IFNs: 3 IFN molecules
– IL-28A, IL-28B, and IL-29
– Co-produced with IFN-β
– But act by binding to a different receptor from
type I IFN receptors

INTERFERON-GAMMA
History and biology

Interferon-gamma (IFN-γ)
• Sole type II IFN
• Made primarily by T cells and NK cells
• More of an interleukin than an interferon?
– Modest antiviral activity
– Prominent derivation from T lymphocytes
– Wide-ranging functions
• Play roles in cellular and allergic immunity
J W Steinke, et al., Middleton’s Allergy 8th edition, 2013, 65-82.

IFN-γ: General history
A. Billiau, P. Matthys Cytokine & Growth Factor Reviews 20 (2009) 97–113
1965 Induction of an IFN activity in human PBMC by phytohemagglutinin
1966 Ag-specific induction of IFN activity during virus infections
1972 New IFN was named ‘immune IFN’
1973 IFN produced during DTH reactions  named ‘Type II IFN’
1980 Nomenclature Committee: definitive name ‘IFN-γ’
1982 Dimeric structure of IFN- γ suggested,
Cloning of IFN- γ from cDNA

IFN-γ: Structure
• Dimeric in solution
• Each subunit
– 6 α-helices, that comprise
62% of the structure
– No β-sheet
– Composed of 140 amino
acids
– No homology with type I
IFNs
S E. Ealick, et al., Science, New Series, Vol. 252, No. 5006 (May 3, 1991), pp. 698-702

IFN-γ: Sources
• During innate immune responses
– Natural killer (NK) cells
– Natural killer T (NKT) cells
– Macrophages
– Dendritic cells

IFN-γ: Sources
• In adaptive immunity
– CD8+ T cells
– Control of infection,
– CD4+ T helper 1 (Th1) subset
• Promotes inflammatory responses
• Clearance of intracellular pathogens
• Class-switching to IgG2a, IgG2b, and IgG3

IFN-γ: Gene
• Human chromosome 12
• Cytogenetic Location: 12q14
• Molecular Location on chromosome 12:
– Base pairs 68,154,769 to 68,159,740
National Library of Medicine (US). Genetics Home Reference [Internet]. Bethesda (MD): The Library; 2013 Sep 16 [cited 2014 April 3]. Available from:
http://ghr.nlm.nih.gov/gene/IFNG.

IFN-γ: Expression regulation
• In innate immunity
– IFN-γ production response to constitutive
expression of transcription factors
• Eomes and T-bet (NK cells)
• T-bet (NKT)
– These transcription factors bind to regulatory
elements that are already accessible within
the Ifng locus, leading to activation of Ifng
transcription

• In adaptive immunity
– Expression by CD4+and CD8+ T cells
– Differentiation process of naïve CD4+ or
CD8+ T cells to Th1 or cytotoxic T effector
cells requires:
• T cell receptor (TCR) stimulation
• Multiple rounds of cell division
• Induction of T-bet
• Epigenetic modifications within the Ifng gene

• Epigenetic regulation: long non-coding
RNAs (lncRNAs) Tmevpg1 (also known as
NeST)
– Positively contribute to IFN-γ production by
CD4+ and CD8+ T cells
– Tmevpg1 is adjacent to the Ifng gene
– Encoded on the DNA strand opposite to that
coding IFN-γ

• Epigenetic regulation: long non-coding
RNAs (lncRNAs) Tmevpg1 (also known as
NeST)
– Tmevpg1 transcription is dependent upon
transcription factors, Stat4 and T-bet
• Which also influence Ifng transcription in CD4+
Th1 T cells
– Tmevpg1 transgenic mice:
• Increased IFN-γ
• Immune to salmonella infection

IFN-γ: Signalling pathways
X Hu, et al., Immunity 31, October 16, 2009, 539-550.

• IFN-γ enhances TLR-
induced TNF production
by disrupting an IL-10-
mediated inhibitory loop
• Increased activity of GSK3
• Negatively regulates IL-10
expression by
suppressing activation of
transcription factors CREB
and AP-1

• IFN-γ enhances TLR-induced
IL-6 and IL-12 production
• Disrupts inhibitory loop mediated
by Notch target genes Hes1 and
Hey1
• Downregulates intracellular
NICD2 amounts
• Inhibits expression of Hes1 and
Hey1

• Via regulation of IL-1R and TLR signaling
• Inhibits IL-1 signaling in macrophages by
downregulating IL-1RI expression
• Blocks induction of MMP downstream of TLR
signaling by
• Superinduce transcription repressor ATF3
• Inhibit transcription activators CREB and
AP-1
• Inhibits CREB activity by suppressing its serine
phosphorylation
• Inhibits AP-1 by downregulating nuclear protein
levels of its subunits

• Inhibits osteoclastogenesis and
bone resorption
• Suppress expression and signal
transduction of RANK, CSF-1R, and
TREM2
• (Receptors critical for the process of
osteoclastogenesis)

• Attenuates fibrosis by:
• Suppresses TGF-βR signaling by
• Induction of inhibitory SMAD (SMAD7)
• Direct inhibition of SMAD3 by STAT1
• Inhibits IL-4R signaling by induction of SOCS1

• In Th1 cell differentiation
• IFN-γ-STAT1 signaling is critical for induction
of T-bet and thus for sustaining the positive
feedback loop
• Leads to heightened production of IFN-γ.

• IFN-γ blocks Th2 cell differentiation
• By inhibiting IL-4-STAT6 signaling

• IFN-γ and STAT1 can block Th17 cell
differentiation
• Mechanism of action is not clear
• Possibly suppresses Th17 cell by targeting
STAT3 (shown by dotted lines)
• Inhibit aryl hydrocarbon nuclear receptor
(AHR)
• Suppression of TGF-β and IL-1 signaling by
IFN-γ may contributes to inhibition of Th17
cell differentiation (not depicted)

• Regulates Treg cell differentiation and
function.
• Block TGFβ-mediated Treg cell
differentiation
• Upregulates expression of T-bet in Foxp3+
Treg cells
• Promotes expression of CXCR3 that
regulates homing of T-bet+ FoxP3+ Treg
cells to sites of Th1 cell inflammation

INTERFERON-GAMMA
Roles with other immune components

IFN-γ and macrophages
1969 Lymphocyte-mediated activation of macrophagesMIF responsible?
1979 Several macrophage-activating factor (MAF) assays established
1983 Anti-IFN-γ antibody neutralizes MAF preparations
1985 Cloned IFN-γ possesses MAF activity

• Driving differentiation from inactive
monocytes into potent effector M1
activated macrophages
– Enhanced adherence, phagocytosis,
degranulation, and production of reactive
oxygen and nitrogen molecules
• Responsible for their accumulation at the
site of CMIR as cells newly capable of
killing intracellular pathogens and cancers

• Activated M1 macrophages
– Induced by IFN-γ
– High producers of IL-1β, TNF-α, IL-6, IL-12,
and IL-23
– but not IL-10
– =>Proinflammatory and participate in Th1
polarization

IFN-γ and NK cells
1980 IFN-γ upregulates NK activity
1983 IL-2 may induce IFN-γ in NK cells
1993 Novel IFN-γ-coinducing factor  IGIF/IL-18
1995 IL-18 induces IFN-γ in NK cells
1991 IL-12 induces IFN-γ in NK cells

IFN-γ and NK cells+PMN
• Stimulates killing by NK cells and
neutrophils
• Stimulates adherence of leukocytes to
endothelial cells through induction of
ICAM-1

IFN-γ and APCs + DCs
1982 IFN-γ enhances MHC Class II expression in mononuclear phagocytes
1984 IFN-γ induces enzymatic breakdown of tryptophan
2000 IFN-γ optimizes IL-12 production by DCs
IFN-γ-induced IDO conditions DCs to
become tolerogenic
1990 IFN-γ induces IDO in vivo
1996 IFN -γ enhances MHC Class II expression in DCs

IFN-γ and APCs + DCs
• Directly stimulates Ag processing
• Stimulates antigen presentation via
increased MHC class I and II expression
• Stimulates cytokine production

IFN-γ and Treg cells
1995 CD4+CD25+ Treg cells defined
2005 Defective functioning of Treg cells in IFN -γR KO mice with
collagen-induced arthritis (CIA)
2006 IFN-γ can convert CD4+CD25 cells into CD4+ Treg cells able to
suppress experimental autoimmune encephalomyelitis (EAE)
Release of IFN-γ by Treg cells

IFN-γ and T-helper cells
1973 T cell-replacing factor (TRF, T cell help for B cells) described
1977 Type II IFN inhibits antibody production in vitro
2005 Th17 cell lineage defined in mice
IFN-γ inhibits differentiation of Th17 cells
and IL-17 production by activated Th
memory cells
1984 IFN-γ proposed as a TRF
1988 Th1 and Th2 clones described
Role of IFN-g in Th1/Th2 paradigm

INTERFERON-GAMMA
Roles in infection defense

IFN-γ and infection defense
• Most important in vivo role
• Establishing effective response towards
pathogens whose elimination from the
body depends on phagocytosis and
intracellular killing
• Weakly inhibits viral replication

Pathogen entry
NK cells produce IFN-γ  primes mononuclear phagocytes
for production of monokines:
TNF-a and IL-12
IFN-γ & TNF-a augment bacteriostatic potential of phagocytes
Guided by IL-12, Th1 response is mounted
Additional IFN-γ production by activated CD4+ and CD8+
T cells

• In Listeria infection, IFN-γ can:
– Augment normal resistance
– Restore compromised resistance
– Rx with neutralizing Ab to IFN-γ abrogated
resistance
– IFN-γ production during the first 2 days of
infection was critical for development of
protective Ag-specific T cells

• In Mycobacterial infections
– Application of IFN-γ on skin lesions of
lepromatous leprosy patients caused
increased infiltration with lymphocytes and
reduction in the local bacterial load
– Mice with a disrupted gene for the IFN-γ
receptor were found to fail controlling infection
with M. bovis.

• Mycobacterial infections in IFN-γ deficient
mice
– Unable to control sublethal doses M.
tuberculosis or M. bovis
– Bacteria multiplied more extensively and
caused more widespread damage in affected
tissues.
– Compromised in both innate resistance in
early phase of infection, and also later
development of protective immunity

IFN-γ and infection defence
• Important factor in directing the immune
response towards the Th1 pathway
• Mitigates excessive extramedullary
myelopoiesis
• Responsible for apoptosis of CD4+ T cells
in the later phases of the immune
response to mycobacteria
• Responsible for the appearance of
‘immunosuppressive’ macrophages

INTERFERON-GAMMA
Roles in autoimmunity

IFN-γ and autoimmunity
• Mechanism is remain unclear
• Epidermal transgenic expression of IFN-γ
leads to
– Anti-dsDNA,
– Anti-histone autoAb
– Glomerulonephritis
• Transgenic IFN-γ expression in other sites
does not lead to systemic autoimmunity??

• Possibly supporting evidence
– Ability of IFN-γ to
• Promote B cell IgG class switching to more
pathogenic autoAb
• Activation of IgG Fc receptors and complement
• Contributes to disease severity
– In end organ damage, infiltration of IFN-γ
secreting T cells resulting in macrophage
activation, inflammation, and tissue damage

• Possibly supporting evidence
– Mutated mice with reduced decay of IFN-γ
mRNA
• Increased IFN-γ signaling and accumulation of
follicular helper T (Tfh) cells
• Increased germinal center B cells and autoAb
• IFN -γR-deficiency in these mice can prevent the
development of lupus

• Possibly contradict evidence
– The experimental autoimmune diseases,
EAE, EAU and CIA, = Th17-driven
– Conditions for optimal in vitro induction of
naive T cells differentiation into Th17 cells by
IL-23 were found to include neutralization of
endogenous IFN-γ

– Ablation of IFN-γ resulted in increased
numbers of IL-17-producing T cells
– Conclusion: Endogenous IFN-γ inhibits
differentiation of Th17 cells
– Ablation of endogenous IFN-g should boost
disease in EAE and CIA.

– Blocking of autoimmune diseases can be
done by injection of syngeneic Treg cells
– In vitro treatment of CD4+CD25 cells with
IFN-γ cause their conversion into CD4+ Treg
cells
– Evidence exists for induced Treg cells to
rapidly release IFN-γ, that may be important
for their suppressive activity

IFN-γ as a therapeutic target?
• Fontolizumab,
– Humanized monoclonal Ab against IFN-γ
– Showed some efficacy in patients with
Crohn’s disease
– Phase II clinical trial investigating its use in
rheumatoid arthritis was terminated because
the first phase did not meet the endpoint

• Amgen’s AMG 811
– Human monoclonal Ab
– Being evaluated in safety trials with subjects
with DLE and subjects with SLE with and
without glomerulonephritis

• Non-specific targeting of IFN-γ
– Impact both innate and adaptive immunity
– Deficiency of IFN-γ is associated with severe
infection
• Targeting to cellular components
regulating IFN-γ expression, such as
lncRNA Tmevpg1, may provide greater
therapeutic benefit without adverse effect
on responses to infection

INTERFERON-GAMMA
Roles in allergy and hypersensitivity

IFN-γ and allergy
• Allergic inflammatory tissue has prominent
presence of IFN-γ
• IFN-γ exacerbates allergic inflammation
through its ability to
– Activate accessory cell function
– Stimulate cytokine secretion
– Induce adhesion molecule expression
– Activate eosinophils and neutrophils

IFN-γ and allergy
• IFN-γ promotes allergic inflammation
– IFN-γ–producing Th1 lymphocytes exacerbate
murine asthma
– Th1-like processes are particularly prevalent
in patients with severe asthma, especially
those with irreversible obstruction and
neutrophilic inflammation

IFN-γ and allergy
• It is frequently stated that the immune
response to allergens in non-allergic
subjects is characterized by Th1-like
lymphocyte responses
• …But without CMIR and cellular
inflammation

IFN-γ and DTH
• Exogenous IFN-γ was found to reverse
inhibition of the DTH response by anti-
CD4 or anti-IL-2R Ab in mouse model
•  supporting the concept that production of
IFN-γ by TH1 cells is essential for the reaction
• There was report of IFN-γ potentiates
contact sensitivity

IFN-γ and DTH
• There were ambiguity in reports analyzing
the role of IFN-γ in DTH reactions
• This reflects the pathogenic complexity of the
systems under study
• Effects may differ depending on
• Ag used (protein or hapten)
• Route of exposure (injection or contact with
the skin)
• Time point during the reaction (during the
sensitization or the elicitation phase)

IFN-γ and DTH
• DTH reactions rely on both natural and
acquired immune response mechanisms
• IFN-γ may act differently on these
components

IFN-γ and Shwartzman reaction
• 2 varients:
• Localized
• Generalized
• Both are two-stage phenomena
• Preparative (sensitizing) injection of
endotoxin
• Eliciting (provoking) injection followed after
about 24 h

IFN-γ and Shwartzman reaction
• Example of human model:
• Thrombohemorrhagic shock that sometimes
occurs in humans with meningococcal sepsis
• In mouse model with Pre-treatment with
neutralizing anti-IFN-γ
• Completely protected against this reaction
• Reduced production of circulating TNF
following the eliciting dose

Take home message
• IFN-γ is the only type II IFN
• It’s a cytokine that is critical for innate and
adaptive immunity
• Its action mainly via JAK-STAT pathway
• It has multifaceted roles: Infection defense
(esp. intracellular pathogens) , CMIR,
autoimmunity, allergy and hypersensitivity

Interferon-gamma and immune system

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