immune effector modules: t cells activate discrete cell

Immune effector modules: T cells activate discrete cell populations

Facultative and obligate intracellular organisms

Extracellular bacteria and fungi

Helminths and biting insects

Viruses

Obligate intracellular organisms

Bypass barriers - insects vectors, animal bites, trauma, ulcerations

Exploit mucosal M cells

Co-evolution with receptors drives narrow host specificity

Viremia needed to seed organs required for transmission - kidneys (urine), skin, salivary glands (secretions), digestive tract (feces)

Viruses

Flavors: ssRNA, dsRNA, DNA

Encoded within virally encoded capsid proteins

Enveloped or not

Classes: Lytic (cytopathic) (polio, flu) versus nonlytic (hepatitis B, LCMV)

Latency: special property of some lytic viruses

Viral Life Cycle

1. Breach barriers

2. Disseminate via lymph nodes

3. Viremia to seed target organs

4. Shedding to new hosts

Immune cells make good targets…

Key players: interferons and its transcription factors

Type 1 interferons: Interferon-/Interferon- (14)

Type 2 interferon: Interferon-

Hybrid interferons: Interferon- (3) {IL-28A, IL-28B,

IL-29}

Auto-enforcing loop: IRF-3 > IFN > Stat1/2 + IRF-9 > IRF7 > IFN’s

Amplification by Type 1 interferons

IRF-3

IRF-7

IFNAR

Stat 1,2,4

IRF-3IRF-7

IFN-

IFN-

IFN

IFN-

IFN

IFN-

Amplification by Type 1 interferons

IRF-3

IRF-7

IFNAR

Stat 1,2,4

IRF-3IRF-7

RNAseL

Anti-Viral State

PKR

IFN

IFN-

IFN

IFNa

IFN

IFN-

IFN-

Human Stat1-deficiency: lethal viral infection

IRF-3

PIRF-3

CBP/p300

NF-B

IRS-7

P

IRS-7

PKR OAS

ISG15ISG54IP-10

iNOS

IRF-E

GAS

PRD NF-B

PRD-LE

ISRE ISRE

Tyk2JAK1

Stat2Stat1

Stat1

Stat1

Stat 2

Stat1

IRF-9

P

P

P

P

Auto-amplification in the Type 1 interferon response

IRF-1

IFN-

IFN-

TRIF(TICAM-1)/TRAM: Anti-viral TLR Adapters

MyD88 MyD88Mal/

TIRAPTRIF

MyD88

NF-B JNK NF-B JNK AP-1 IRF-3

Interferons, RANTESIL-1, TNF, IL-6, IL-8, antimicrobial peptides

TLR 1, 2, 4, 6 TLR 3, 7, 9 TLR 4TLR 5, 7, 9

TRAM

MyD88

Defects in UNC-93B abolish TLR3, 7, -8, -9 signaling against viruses.

Abolishes cross-priming.

Human mutations in UNC-93B and dominant-negative TLR3 associated with HSV encephalitis.

Cytosolic dsRNA detectors - RNA helicases

Cell 122:645-7, 2005

dsRNA (5’-triP-ssRNA for flu) binds cytosolic RIG-I and/or Mda5, exposes CARD domain, binds MAVS/Cardif/IPS-1, activates kinase complexes leading to phosphorylation of IRF-3/IRF-7 and IB. MAVS targeted by HCV.

Seth RB et al., Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-B and IRF3. Cell 122:669-82, 2005.Kawai T et al., IPS-1, an adaptor triggering RIG-1 and Mda5-mediated type 1 interferon induction. Nature Immunol 6:981-8, 2005.Meylan E et al., Cardif is an adaptor protein in the RIG-1 antiviral pathway and is targeted by hepatitis C virus. Nature 437:1167-72.

NOTE: Dispensable in plasmacytoid DCs

Cytosolic RNA/DNA recognition pathways

DNA::DAI

(DNA-dependent activator of IFN-regulatory factors)

TBK1::IRF3

IFN-

Induced Synthesis

2-5(A) Synthetase

ATP 2-5(A)

dsRNA

RNaseL RnaseL

(inactive) (active)

AUG AAAAA

mRNA Degraded mRNA

Inhibition of Protein Synthesis

PKR, phosphorylated

(active)

dsRNA-dependent protein kinase, PKR

(inactive)

dsRNA+ ATP

eIF2 , eIF2 Phosphorylated

ATP

eIF2-GDP, phosphorylated

eIF2-GTP, Phosphorylated

GEF

IFNAR

KO with increased viral susceptibility

IFN activates anti-viral cellular miRNAs

Activation by IFN is time- and dose-dependent…

Expression of IFN-inducible miRNAs inhibits viral replication…

Intriguing Cell Biology - Utilization of MVBs by Viruses

Plasmacytoid DC (pre-DC2)

Produce early IFN/ after incubation of PBMC with virus (independent of RNA helicase

pathway)

Prevalence: 0.1-0.3% PBMC

Phenotype: CD4+, CD11c-, IL3R+, CD62L+

Biology: Migrate to HEV and protect transiting naïve

lymphocytes

TLRs activate the virus recognition response early in pDCs

Plasmacytoid DC

pDCs use components of autophagy pathway for TLR7 ssRNA detection

Anti-viral Cytokines

Type 1 Interferons Anti-viral

NK cytotoxicity

T cell survival, DC maturation

IL-6 Systemic response

Cell recruitment

IL-12 Type 1 immunity

NK cell activation

IL-15 CTL and NK growth,

survival

NK Cells and Anti-Viral Host Immunity

Mechanism

IFN-/ Cytotoxicity, Anti-viral

IL-12 Cytokines

(IFN-, TNF, LT, TRAIL, TWEAK)

Antibody ADCC

Evidence

NK-deficient human (severe 1o HSV, VZV, CMV infections)

Mouse MCMV - requires Ly49H

Duncan’s syndrome (X-linked lymphoproliferative disease)

NK Cells Target Herpesviruses

Genetic Evidence

NK-deficient girl with severe primary herpesvirus infections

Murine klra8 (Ly-49H) deficiency

Unable to clear murine CMV

Human SAP (SLAM-assoc. prot.) deficiency

Loss of functional 2B4 (CD244) NK activating receptor with fatal EBV infections

Direct recognition of murine CMV-encoded proteins by NK receptors

Suggests NK receptor diversity may be driven by herpesviruses…

Direct recognition of murine CMV-encoded proteins by NK receptors

Are NK cells simply antiviral T cells?

NK cell

MCMV-infected cells

Ly49P

H-2Dk/MCMV

Desrosiers M-P et al. Nature Genetics 37:593-99.

SAP

2B4 ?

Duncan’s Syndrome

EBV-infected cell

NK Cell

SAP

2B4 ?

EBV-infected cell

Early death due to primary progressive EBV infection. Mutation in SAP (SLAM-associated protein), an X-linked adapter protein, or rarely in XIAP

(inhibitor of apoptosis).

Mutant SAP transduces negative instead of positive signals from engaged 2B4 receptors.

NK Cells Localize Anti-Viral Immunity

Liver

MCMV

IFN-

IFN-

MIP-1

Mig

CD4 CD4

CD4

NK NK

Major Viral Effectors: CD8+ CTL

% IFN

( )

Time (Hrs)

0 5 10

Infectious Virions 3-10 Hrs.

cytolysis

cytolysis

Virus peptide Remove peptide Replace peptide

Dogma:

Non-cytopath. virus: CTL

Cytopath. Virus: Ab

CD8 Response to LCMV

Clonal Apoptosis Memory Burst Size

Burst (IFN) (perforin, IL-15, antigen)

All Tetramer-Positive CTL Have Effector Function

No Bystander

Peptide-specific Activation

Antibodies: Prevent Re-infection

LCMV

Can’t maintain CD8’s without CD4 Help

Viruses attack common cellular defense pathways

Viruses block activation

of cellular apoptosis

pathways

CMV attacks MHC class I pathways at multiple levels

Large DNA viruses

(herpesviruses, poxviruses)

encode additional proteins to

mitigate host defense and

sustain infectivity

Induction of MIC-A at foci of CMV in infected lungs

MIC-A

CMV

cytolyticresponse

8CD (-)

-A restricted

Target

+ -orMICA

T cell

C R

CMVpeptide

NKG2D enhances cytolytic activity after TCR engagement

IFN-

IL-2 IL-4

cytokinesecretion

8CD (-)

-A restricted

Stim

+ -orMICA

T cell

CMVpeptide

TNF

Decreasing Peptide

Joe Bob Briggs:

“…three dead bodies, two dead birds, multiple seagull divebomb attacks, playground crow attack, bird migration, bird flocking, exploding gas station, two car crashes, crow kung-fu, kamikaze seagull…”

4 Stars. A classic. Check it

out!

Influenza - Obligate Virology

Orthomyxovirus Negative-sense ssRNA, eight-segmented genome

Types A (avian, humans, responsible for pandemics), B (avian, humans, seals), C (avian, pigs, humans rare)

10 proteins:

PB1(-F2), PB2, PA Heterotrimeric polymerase (?mitochondrial apoptosis)

HA Homotrimeric binding and fusion element

NA* Homotetrameric enzymatic release factor

NP Nucleoprotein (nucleocapsid packaging)

M1 Transport of viral RNPs

M2^ Homotetrameric cation channel pore

NS1 Binds RNA; interdicts host translational machinery and defense (PKR,

cytokines)

NS2 Nuclear export of viral RNPs

* Neuraminidase inhibitor target

^Amantadine target

Relevant Life Cycle Issues

1. An intestinal infection of wild waterfowl.

2. Crosses to mammals through close contact.

3. Multiple ‘crosses’ enhance capacity to establish

mutants and reassortment variants

adapted to mammalian hosts.

4. HA species specificity: sialic acid -2,3 galactose linkage (avian intestine)

sialic acid -2,6 galactose linkage (human trachea)

both (pig trachea)

5. NA compatibility: human viruses gain -2,6 activity

stalk length (longer NA enhances activity in humans)

6. HA, NA Adaptations HA glycosylation; HA1/HA2 fusion domain (expanded basic amino acid repeat in highly pathogenic chicken H5/H7/H9 flu -HPAI- enhances spectrum of proteases that can activate HA fusion event; may explain pathogenicity of co-infection with bacteria)

Mutation and reassortment drive influenza A epidemics and pandemics

Live chickens and ducks in same cages

Asian Live-Animal Markets -

The Great Zoonotic Mixer

Influenza Pandemics

Year Common Name Subtype OriginDeaths

1889 - H2N2 ?Europe 6 million

1898 - H3N2 ?Europe 0.5 million

1918 Spanish Flu H1N1* ?Eurasia 40 million

1957 Asian Flu H2N2* China 4 million

1968^ Hong Kong Flu H3N2* China 2 million

1977^ Russian Flu H1N1+ China/Russia ?* Contained elements from avian viruses

+ Laboratory-derived from frozen stock (persons pre-’50s immune)

^Antigenic variants continue to co-circulate

Relevant Immunology

Innate immunity: type 1 IFNs, TNF-, Mx proteins

HA antibodies: Neutralize infectivity, protective

NA antibodies: Restrict viral spread

Cytotoxic CD8 T cells: M2, PB2, HA, NP specificity common M2 specificity almost universal

CD8 TCR / chains

V17/V10.2

HLA-A2 (A*0201)

Influenza A Matrix Protein amino acids 58-66

Stewart-Jones et al. Nature Immunol 7:657, 2003

The Most Common Human TCR in the World

Treanor, NEJM 350:218, 2004

HA1 A/Panama/2007/99 HA1 A/Fujian/411/2002

Antigenic Drift - 2003/04

Influenza NS1 protein sequesters

viral ss RNA to block cellular anti-

viral defense

Why do they die?…

Verified H5N1 influenza through October 2006

Human deaths/cases = 152/256 (59%)

HIV

Worldwide: 42 million infected

29 million dead

14,000 new infections/day

2/3 infected persons in Africa

U.S.: ~1 million infected including 400,000 dead

(appeared 1983)

Worldwide Estimates of Numbers of HIV-Infected Persons

HIV Origins - Primate Lentiviruses

SIVcpz - West equatorial Africa = M group

Cameroon = N group

Gabon = O group

HIV-2 = SIVsm (sooty mangabey)

Infection/Disease in areas of active bushmeat trade.

HIV Origins

SIVcpz - Asymptomatic infection of chimpanzees (up to 1% in areas of west Central Africa)

HIV-1: M group consists of 11 clades

Last common ancestor entered human population around 1930 (+ 20 yrs)

Prevalent HIV Clades

HIV is a primate lentivirus

Lentiviruses can infect nondividing cells

Replication driven from long terminal repeats

Structural genes - gag, pol, env

Regulatory genes - tat, rev

Accessory genes - vif, vpr, vpu, nef

HIV life-cycle

TRIM5

APOBEC

HIV vif sequesters

APOBEC enzymes from

the budding virions

HIV Pathogenesis

M

DC-SIGN

1. Entry at sites of M cells or trauma (STDs)

2. Transit to LN via C-type lectins* on dendritic cells

3. Peak CD4+ T cell infection days 4-7

4. Viremia peaks day 14

5. All lymphoid tissues infected by day 23

*DC-SIGN, MR, Langerin

HIV infection occurs predominantly at mucosa

Dendritic cells mediate transit of virus to regional lymph nodes via CLRs

Massive loss of mucosa-associated lymphocytes of the small intestine precedes systemic CD4 T cell loss

HIV Receptors

CD4

R5

X4

1o Infection: M-tropic, CCR5

Turnover 1010 virions/day

Progressive CD4 T cell destruction

CXCR4

T-tropic

Syncytium-forming

Natural History of Untreated HIV Infection

HIV Resistance

1. CCR532 - slow progression if infected

20% W. European Caucasians Heterozygous

1% Homozygous

2. HLA class I homozygosity - rapid progression

3. Rare HLA class I alleles - slow progression (suggests virus near mutational threshold)

SIV DNA Vaccine (gag/env + IL-2)

Rhesus

Lethal SIV Challenge

Day 0 Day 14 Day 70

CTL V 0.2-0.4% 18-40%

C 0 1-4%

Neutralizing Ab V 0 Equivalent

C 0 Equivalent Depressed with CD4

Virus V 106-107 <103

C 107-108 105-106

Outcome V All alive with normal CD4s

C 50% die, all with loss of CD4s

140 days

Barouch et al., Science 290:486, 2000

Caveat: CTL escape mutants

Why no HIV vaccine?

1. Escape variants/altered peptide ligands - virus operates near mutational threshold

2. Neutralizing antibodies low-affinity, arise late (conformationally hidden, glycan shielding, mutational escape, evolutionary escape from ‘natural antibodies’, polyclonal B cell activation may impede)

3. Loss of CD4 help required for CD8, antibody responses

4. Immune exhaustion with PD-1 expression on CD4 and CD8 anti-HIV T cells