NK mediated Antibody Dependent Cellular Cytotoxicity in HIV infections Amy Chung Dr. Ivan Stratov Prof. Stephen Kent
ADCC process consists of Target cell QuickTime and a TIFF (Uncompressed) FcγR decompressor are needed to see this picture. IgG Ab Ag 1. Target cells expressing pathogen s antigen on the surface
ADCC process consists of Target cell QuickTime and a TIFF (Uncompressed) FcγR decompressor are needed to see this picture. IgG Ab IgG Ab Ag 1. target cells expressing pathogen s antigen on the surface 2. Antibodies of IgG isotype to target antigen
ADCC process consists of Target cell IgG QuickTime FcγR and a TIFF (Uncompressed) FcγR decompressor are needed to see this picture. IgG Ab Ag 1. target cells expressing pathogen s antigen on the surface 2. Antibodies of IgG isotype to target antigen 3. Effector cells (NK cells) with FcγR (gamma receptor)
ADCC process consists of QuickTime and a TIFF (Uncompressed) FcγR decompressor are needed to see this picture. Ag 1. target cells expressing pathogen s antigen on the surface 2. Antibodies of IgG isotype to target antigen 3. Effector cells (NK cells) with FcγR (gamma receptor) Collaboration between innate (NK cells) and adaptive (Ab) immune response NK cells kill cells in the absence of MHC while having high specificity mediated by Antibodies
Importance of ADCC against HIV HIV Human Cohort studies: Low ADCC titres correlate with progression and CD4+ cell decline Baum 1996, Forthal 21, Macaque Cohort Studies: Animals with higher ADCC responses progress more slowly Banks 22, Ohkawa 1994 Macaque Vaccine Studies: Vaccine elicited ADCC activity correlates with reduced viral load Gomez-Roman 25, Florese 26, Monkey Passive Transfer Study: Abrogating Fc binding substantially reduces efficacy of Nab Hessell 27
Novel ICS Assay to detect ADCC HIV positive human whole blood + gp14 protein or overlapping HIV peptide pools (15 aa overlapping by 11 aa) gp14 or HIV Peptide pools: 15aa overlapping by 11aa Brefeldin A Incubate 37º 5% CO 2 HIV +ve whole blood Stain for CD2, CD56, CD3 Stain for intracellular IFNγ Acquire cells on FACS.95 2.4 SSC 26.1 Lymphocytes Cell Counts CD3- Lymphocytes 44.2 IFNγ 33.5 45.1 CD56+ CD3- (Natural Killer) cells expressing IFNγ FSC CD3 CD56
IgG mediates NK cell activation in ICS Assay to detect ADCC HIV serum mediated Specifically IgG mediated Not complement mediated Does not require co-stimulation Readily maps linear ADCC epitopes Can phenotype response Measures multiple effector functions Gated on CD3- lymphocytes Autologous cells + autologous plasma Autologous cells + donor plasma Donor cells + donor plasma Donor cells + autologous plasma 23.7%.%.% 22.8% IFNγ CD2 Stratov, Chung, Kent, J Virol. 28
Phenotype of NK effector cells in ADCC ICS Assay Cell Surface Marker Expression Effector Molecule Expression CD3 CD4 CD8 T cell markers TCRαβ TCRγδ CD19 B cell CD2 markers CD2 + CD16 +/ CD56 +/ CD85j NK cell +/ CD94 (KLRD1) markers +/ CD158a (KIR2DL2) +/ CD161 (KLRB1) +/ NKB1 (KIR3DL1) +/ IFNγ + Granzyme + Perforin + TNFα + CD17a + IL-2 IL-4 Effector cells express characteristic NK cell phenotype
Cytolytic functions of NK cells measured by ADCC ICS Assay DMSO ENV peptide pool gp14.4 1. 7.2 CD17a IFNγ CD56.3 1.9 6.8 CD56
Cytolytic functions of NK cells measured by ADCC ICS Assay DMSO HIV CD17a IFNγ DMSO ENV peptide pool.4 1. CD56.3 1.9 gp14 7.2 6.8 Granzyme B Perforin CD17a 1. 6.8 CD56 CD17a.4 2.8
Recruit ADCC Cohort Currently recruited 8 ART naïve subjects ADCC responses measured by ICS Assay Protein pool GAG POL ENV RTV VVN ANY HIV No. of Cohort 13 46 53 17 27 7 % of Cohort 16% 55% 66% 21% 34% 87% 8 7 6 5 4 3 2 1 GAG POL ENV RTV VVN ANY HIV Protein Peptide Pools RTV= Rev, Tat, Vpu VVN= Vif, Vpr, Nef combined peptide pools
What epitopes do ADCC target? Use of HIV overlapping peptide pools allows us to readily map linear ADCC epitopes DMSO ENV peptide pool ENV peptides 61-9 ENV V3 peptide 78 ENV peptides 121-15.85.63 5.47.49 3.88.98 4.23.14.46 IFNγ 98.6 1.28 72.5 21.4 8.4 15.2 71.5 23.3 95.2 4.19 CD17a
What epitopes do ADCC target? Use of HIV overlapping peptide pools allows us to readily map linear ADCC epitopes DMSO ENV peptide pool ENV peptides 61-9 ENV V3 peptide 78 ENV peptides 121-15.85.63 5.47.49 3.88.98 4.23.14.46 98.6 1.28 72.5 21.4 8.4 15.2 71.5 23.3 95.2 4.19 gp14 protein V3 deleted gp14.3.74.25.11 IFNγ 92 6.97 96.7 2.96 CD17a
Mapping ADCC epitopes on ENV cleavage C1 V1 V2 C2 V3 C3 V4 C4 V5 C5 C6 C7 gp12 gp41 Identified ADCC epitope Variable region Constant region Signal Peptide Figure: Map of identified ADCC Epitopes in gp16 Envelope glycoprotein Identified 37 novel ENV specific linear ADCC epitopes 9 epitopes shared by 2 or more subjects Additional epitope in VPU mapped Currently mapping additional Pol epitopes
Do these ADCC responses force immune escape? Investigate the possibility of viral mutational escape from ADCC activity See if ADCC exerts significant immune pressure upon the virus
Viral sequence variation at ADCC epitopes 29% 29% 4% 4% 4% 4% 4% 4% 17% 17% 21% 21% Investigated biological relevance of mapped ADCC epitopes Cloned and sequenced several subjects concurrent autologous plasma virus samples across the mapped ADCC epitopes and identified variations from HIV-1 consensus sequence 21% 21% Identified sequence variation in 96% of ADCC epitopes 1 2 3 4 5 6 Number of amino acid changes within epitope sequence in autologous native virus compared to consensus B HIV
ADCC escape 5 4.5 4 A 25 2 C 3.5 3 15 2.5 2 1 1.5 1 5.5 1ug 1ng 1ng 3ng 1ng.3ng 3ug 1ug 3ng 1ng 3ng 1ng VPU V19 VPU I19 VPU A19 Consensus Native % of IFNγ secreting cells 12 1 8 6 4 2 3ug 1ug 3ng 1ng 3ng 1ng B Consensus Native Concentration of peptide per 2ul well
ADCC escape 5 4.5 4 A 25 2 C 3.5 3 15 2.5 2 1 1.5 1 5.5 1ug 1ng 1ng 3ng 1ng.3ng 3ug 1ug 3ng 1ng 3ng 1ng VPU V19 VPU I19 VPU A19 Consensus Native % of IFNγ secreting cells 12 1 8 6 4 2 3ug 1ug 3ng 1ng 3ng 1ng Consensus Native B 4 3.5 3 2.5 2 1.5 1.5 3ug 1ug 3ng 1ng 3ng 1ng Consensus Native D Concentration of peptide per 2ul well
Future Aims Functionally compare more consensus and autologous ADCC epitopes Conduct a prospective and longitudinal study of ADCC from HIV cohort Identify ADCC epitopes associated with slow progression of disease Reach a clearer understanding of significance of ADCC responses in HIV infection
Stephen Kent Acknowledgments Ivan Stratov Rob Center Erik Rollman Kent Lab QuickTime and a TIFF (LZW) decompressor are needed to see this picture.