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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