Transcript Slides - View the full AIDS 2016 programme

Neutrophils mediate potent and
rapid anti-HIV antibody-dependent
functions
Matthew J Worley, Anthony D Kelleher,
Stephen J Kent and Amy W Chung
The University of Melbourne, Australia
FcR mediated antibody functions
• Antibodies can recruit innate cells via FcR
• NK cells can mediate ADCC responses via an
Fcγ receptor
• Monocytes and neutrophils are able to
mediate phagocytosis and ADCC responses via
Fcγ receptors
FcR mediated antibody functions
Virus
Phagosome
Antibody
Granules
Kill
Fc Receptor
Antigen
Antibody
Fc Receptor
Effector cell
Infected cell
FcR mediated antibody functions in HIV
• High levels of ADCC antibodies correlate with
delayed disease progression with HIV
(Baum et al. 1996, Chung et al. 2011, Wren et al. 2013)
• The RV144 vaccine trial highlighted the importance
of Fc mediated antibody functions
– IgG induced ADCC to envelope protein (Haynes et al. 2012)
– IgG3 to the V1V2 region reduced risk of infection (Yates et al. 2014)
– IgG3 mediated polyfunctional immune responses (Chung et al. 2014)
• NK cells and monocytes have been the main focus of
recent studies
Leukocyte cell prevalence
FcγRIIIB
FcγRI
FcγRIIB
FcγRIIB
FcγRIIA
FcγRIIIA
Neutrophils
~65%
T cells
~20%
NK cells
~5%
B cells
~5%
FcγRIIB
FcγRI
Monocytes
~5%
FcγRIIIA
FcγRIIA
Aim
• Can neutrophils mediate antibody-dependent
anti-HIV responses?
Cohort clinical data
HIV
Number of samples
33
CD4 count entry, cells/μl (median, Range) 566 (330-1156)
Viral load RNA copies/ml (median, Range) 26400 (382-339000)
Neutrophils are able to mediate anti–HIV
antibody-dependent phagocytosis
gp120 protein
Fluorescent bead
Antibody
Fc Receptor
Neutrophil
Phagosome
Phagocytic Score
25
20
15
10
5
0
HIV
25ug/ml IgG
HIV negative
Method adapted from Ackerman et al. 2011
Neutrophils are able to mediate anti–HIV
ADCC responses
Lysis
gp120
protein
Antibody
Fc Receptor
CFSE
Pkh stained viability
membrane stain
Loss of CFSE
viability
stain
%Lysis (Pkh+ CFSE-)
60
40
20
0
HIV
25ug/ml IgG
HIV negative
Method adapted from Gómez-Románet al. 2006
The ADNP and ADCC responses
correlate
%Lysis (Pkh+ CFSE-)
60
r= 0.5749
P= 0.0005
40
20
0
0
5
10
15
Phagocytic score
25ug/ml IgG
20
25
Leukocyte cell prevalence
FcγRIIB
FcγRIIIA
FcγRI
FcγRIIA
FcγRIIB
FcγRIIIB
FcγRI
B cells
~5%
NK cells
~5%
FcγRIIIA
Neutrophils
~65%
Monocytes
~5%
FcγRIIB
FcγRIIA
T cells
~20%
How do neutrophil ADCC responses compare to
other innate cells?
%Lysis (Pkh+ CFSE-)
80
NK cells
60
40
20
0
0
50
Concentration (mg/ml)
100
PBMC’s mediate enhanced levels of ADCC compared
to NK cells at all antibody concentrations
%Lysis (Pkh+ CFSE-)
80
NK cells
PBMCs
60
40
20
0
0
50
Concentration (mg/ml)
100
Neutrophils perform similar levels of ADCC to
PBMC’s
%Lysis (Pkh+ CFSE-)
80
NK cells
PBMCs
Neutrophils
60
40
20
0
0
50
Concentration (mg/ml)
100
Monocytes perform similar levels of ADCC
responses to Neutrophils and PBMC’s
%Lysis (Pkh+ CFSE-)
80
NK cells
PBMCs
Neutrophils
Monocytes
60
40
20
0
0
50
Concentration (mg/ml)
100
How do neutrophil ADCC responses
compare to other innate cells?
%Lysis (Pkh+ CFSE-)
80
NK cells
60
40
20
0
0
1
2
Time (Hours)
3
4
PBMC’s mediate enhanced levels of
ADCC compared to NK cells
%Lysis (Pkh+ CFSE-)
80
NK cells
PBMC
60
40
20
0
0
1
2
Time (Hours)
3
4
Monocytes mediate enhanced levels of
ADCC compared to PBMC’s and NK cells
%Lysis (Pkh+ CFSE-)
80
NK cells
PBMC
60
Monocytes
40
20
0
0
1
2
Time (Hours)
3
4
Neutrophils mediate rapid ADCC
responses to HIV envelope protein
%Lysis (Pkh+ CFSE-)
80
*
*
*
PBMC
60
Monocytes
*
Neutrophils
40
*
20
0
NK cells
0
1
2
Time (Hours)
3
4
Summary and conclusions
• Neutrophils can mediate HIV-specific antibodydependent phagocytosis and ADCC responses
• Neutrophils mediate ADCC responses faster than other
innate effector cells
• Rapid action and high magnitude of ADCC by neutrophils
highlights their potential importance early in HIV
infections
Future directions
• Recruit a new cohort of a progressors and controllers
• Do neutrophils utilise other antibody-dependent
mechanisms to control HIV?
• Do the RV144 vaccinees mediate antibody
dependent functions with neutrophils?
Acknowledgment
Peter Doherty Institute/ University of
Melbourne
Amy Chung
Stephen Kent
Ivan Stratov
Matthew Parsons
Adam Wheatley
Vijaya Madhavi
Hyon-Xhi Tan
Sarah Lloyd
Fernanda Ana-Sosa Batiz
Kevin John Selva
Wen Shi Lee
Hillary Vanderven
Joshua Glass
Anne Kristensen
Vinca Alcantara
Thakshila Amarasena
Kirby Institute/ University of New
South Wales
Anthony Kelleher
Ansari Shaik
Flow cytometry
Tina Luke
Members of the flow core
Funding
National Health and Medical
Research Council