Differential cytokine responsiveness Differential cytokine responsiveness to toll-like receptor (TLR) ligand to toll-like receptor (TLR) ligand

stimulation in HIV-1 resistant sex-stimulation in HIV-1 resistant sex-workers from Nairobi, Kenyaworkers from Nairobi, Kenya

T. Blake Ball Ph.D.T. Blake Ball Ph.D.Department of Medical MicrobiologyDepartment of Medical Microbiology

University of ManitobaUniversity of Manitoba

HIV resistance in the Pumwani HIV resistance in the Pumwani cohort:cohort:

Years of Followup

20181614121086420

Surviv

al P

robability

1.0

.8

.6

.4

.2

0.0

Epidemiological definition of resistance:• active sex worker• >3 years of follow-up• HIV negative by PCR and serology• Clinically no evidence of disease

Reduced incidence of HIV seroconversion with increasing duration of follow-upReduced incidence of HIV seroconversion with increasing duration of follow-up

Modified from Fowke et al.Lancet 1996

Why Does Why Does ResistanceResistance Develop?Develop?

• Resistance to infection by HIV-1 associates with:

• HIV specific cell mediated immune responses,• Compartmentalized HIV specific cell mediated and antibody responses

• Multifactoral immunogenetic factors:• specific HLA alleles (Lacap, Turk, Hardie MOPE0017, MOPE0020, MOPE0024), • polymorphisms in IRF-1 genes (Ji MOAX 0103), • site specific HIV inhibitory factors (Iqbal WEAA 0504)• undescribed factors

Toll-like receptors (TLR) play a critical Toll-like receptors (TLR) play a critical role in innate immunity, recognizing role in innate immunity, recognizing

conserved Pathogen-Associated conserved Pathogen-Associated Molecular PatternsMolecular Patterns

TLRs link innate and adaptive immunity:Regulation of CD4 T cell development

through TLRs

NEJM 2000

We hypothesize that: We hypothesize that:

HIV resistant women have altered HIV resistant women have altered innate responses such that they are innate responses such that they are more likely to develop cell-mediated more likely to develop cell-mediated immune responses to HIV-1.immune responses to HIV-1.

Study design:Study design:

Cross sectional comparison of cytokines known to drive cell mediated (Th1) responses in HIV resistant sex workers, newly enrolled HIV negative sex workers and HIV non-SW controls.

Measure cytokines (IL-12, IFN-, IFN- and IL-10) after short-term PBMC culture (18 hr) in response to ligands for TLR-2 (Staphylococcus aureus), TLR-4 (LPS) and TLR-7 (Imiquamode anolog-3M)

HIV resistant women have elevated IL-10, but HIV resistant women have elevated IL-10, but not IL-12p40, IFN-not IL-12p40, IFN-, nor IFN-, nor IFN- responses to responses to

TLR2 stimulationTLR2 stimulation

10

100

1000

10000

p = 0.006

p = 0.0001

Media SAC (0.004%)

IL-1

0 (

pg

/ml)

10

100

1000

10000

Media SAC (0.004%) SAC (0.001%)

IL-1

2p40

(pg

/ml)

Media SAC (0.001%)

10

100

1000

10000

100000

Media SAC (0.004%) SAC (0.001%)

IFN

- (

pg/m

l)

10

100

1000

10000

Media SAC (0.004%) SAC (0.001%)

IFN

-2a

(pg

/ml)

Media SAC (0.004%)

Media SAC (0.004%)

Media SAC (0.004%)

Media SAC (0.004%)

HIV resistant womenHIV negative SWLR controls

HIV resistant women have elevated IL-10, HIV resistant women have elevated IL-10, depressed IFN-depressed IFN- to TLR4 stimulation to TLR4 stimulation

10

100

1000

10000

Media LPS (0.5 g/ml)

IL-1

2p40

(pg

/ml)

10

100

1000

10000

Media LPS (0.5 g/ml)

p = 0.0001

p = 0.003

IL-1

0 (

pg

/ml)

10

100

1000

10000

Media LPS (0.5 g/ml)

p = 0.021

p = 0.009

IFN

- (

pg/m

l)

HIV resistant womenHIV negative SWLR controls

HIV resistant women have elevated IL-10, HIV resistant women have elevated IL-10, depressed IFN-depressed IFN- to TLR7 stimulation to TLR7 stimulation

10

100

1000

10000

Media 3M (5 g/ml)

IL-1

2p40

(pg

/ml)

10

100

1000

10000

Media 3M (5 g/ml)

p = 0.0019

IL-1

0 (p

g/m

l)

10

100

1000

10000

100000

p = 0.013

p = 0.05

Media 3M (5 g/ml)

IFN

- (p

g/m

l)

10

100

1000

10000

Media 3M (5 g/ml)

IFN

- 2

a (p

g/m

l)

HIV resistant womenHIV negative SWLR controls

ResultsResults

Resistant women have depressed responses Resistant women have depressed responses in cytokines responsible for cell mediated in cytokines responsible for cell mediated immune responses (IFN- immune responses (IFN- ) and elevated ) and elevated immunosuppressive (IL-10) responses. immunosuppressive (IL-10) responses.

Is this a deficit of IFN- Is this a deficit of IFN- production or are production or are the depressed IFN- the depressed IFN- responses simply due responses simply due to immunoregulation by excess IL-10.to immunoregulation by excess IL-10.

We tested the IFN- We tested the IFN- responses to TLR responses to TLR stimulation in the absence of IL-10 (by stimulation in the absence of IL-10 (by blocking IL-10).blocking IL-10).

Does blocking of Does blocking of immunosuppressive IL-10 immunosuppressive IL-10

recover responsiveness of IFN-recover responsiveness of IFN-

Fold IFN- Response to TLR stimulation plus IL-10

0

1

2

3

4

5

6

7

8

9

Fold

IFN

-

Res

pons

e

IFN- Response to TLR stimulation plus IL-10

+ant

i10

+ant

i10

+ant

i10

+ant

i1010

100

1000

10000

100000

IFN

- (

pg/m

l)

Removal of “higher” levels of IL-10 does not restore IFN- levels to those observed in controls.

LPS SAC

HIV resistant women

LR controls

ConclusionsConclusions Resistant women have significantly altered innate Resistant women have significantly altered innate

immune responses to TLR stimulation. immune responses to TLR stimulation.

Altered innate and therefore adaptive immunity Altered innate and therefore adaptive immunity has implications for resistance/susceptibility to has implications for resistance/susceptibility to other infectious and immune mediated diseases.other infectious and immune mediated diseases.

No apparent bias towards generation of cell No apparent bias towards generation of cell mediated responses. Overall, appears to be a mediated responses. Overall, appears to be a suppressed immune response.suppressed immune response.

How could they lead to resistance ? HIV How could they lead to resistance ? HIV resistance may be due to the resistance may be due to the LACKLACK of a response, of a response, rather than the induction of a strong response.rather than the induction of a strong response.

Further Questions….Further Questions….

Why are these women different? Why are these women different? Altered TLR expression, signaling? Altered TLR expression, signaling?

Known TLR 2+4 polymorphisms do not account for this Known TLR 2+4 polymorphisms do not account for this (Marlin TUPDA07)(Marlin TUPDA07)

Several Several correlatescorrelates of HIV resistance have been identified of HIV resistance have been identified Demonstrates that this group is biologically distinctDemonstrates that this group is biologically distinct

How may these associations explain resistance either alone How may these associations explain resistance either alone or in concert? or in concert? Resistance is likely multifactoral and complex. Replication in Resistance is likely multifactoral and complex. Replication in

other HEPS groups critical as is multivariate analysisother HEPS groups critical as is multivariate analysis

Altered TLR signaling relevant to other infectious/immune Altered TLR signaling relevant to other infectious/immune diseases?diseases?

Correlates of resistance

HIVHIV

HIV

HIV

HIV

HIV

systemic compartmentgenital tract

Trappin-2

RANTES

IgA

IRF-1

CTLCTLCTLCTL

CTL

CTL

HIV stim

IL-10TLR stim

CTL

CTLCD3

Thanks to:Thanks to:

The women of the Majengo and Pumwani cohortsThe women of the Majengo and Pumwani cohorts

University of ManitobaUniversity of Manitoba FundingFunding

Dr. Kent HayGlass Dr. Kent HayGlass Dr Frank Plummer Dr Frank Plummer Crystal Marlin Crystal Marlin Rongrong MaoRongrong Mao

University of NairobiUniversity of Nairobi

Dr Charles Wachihi Dr Charles Wachihi Dr Joshua Kimani Dr Joshua Kimani

Colleagues and staff of the WHO collaborative center for STI Colleagues and staff of the WHO collaborative center for STI ResearchResearch