Adenovirus engineered human dendritic cell vaccine induces natural killer cell chemotaxis
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1 Adenovirus engineered human dendritic cell vaccine induces natural killer cell chemotaxis via CXCL8/IL 8 and CXCL10/IP 10 chemokines Lazar Vujanović, Ph.D. Research Instructor P.I. Lisa H. Butterfield, Ph.D.
2 Presenter Disclosure Information Lazar Vujanović, Ph.D. The following relationships exist related to this presentation: No Relationships to Disclose
3 Introduction Dendritic cells (DC) are the most potent antigen presenting cells capable of effective up take, processing, and presentation of antigenic epitopes Natural killer (NK) cells are essential effector cells of the innate immunity that play an important role in antitumor and antimicrobial immune defense DC and NK cell cross talk links innate and adaptive immunity, and plays a key role in host immune responses against infectious agents and tumors IFN γ IL 13 IL 1β TNF α GM CSF NK Cell Activation DC Maturation IL 12p70 IL 23 IL 18 IL 15 IL 2 TNF α IL 10
4 Introduction Utilize first generation ( E1 and E3) recombinant adenoviral vectors (AdV) as vehicles for antigen engineering of DC based tumor vaccines Monocyte derived DC can be efficiently transduced with recombinant adenoviral vectors (Ad.DC) and are safe for clinical trials AdV infection induces an intermediate level of DC maturation (Vujanovic, L. et al. Cancer Immunol Immunother. 2009; 58: ) DC transduced with AdV encoding for a tumor antigen stimulate antigenspecific CD4 + and CD8 + T cell responses
5 Introduction, Cont. Ad.DC effectively activate both CD56 lo CD16 + and CD56 hi CD16 NK cell subsets Ad.DC induce NK cell activation as shown by increased expression of activation marker (CD69), proliferation, IFN γ secretion, tumoricidal activity in vitro, and importantly strong antitumor activity in vivo Ad.DC induced NK cell activation is mediated by cell tocell contact Ad.DC and mdc mediated NK cell activation is mediated by trans presented IL 15 and transmembrane TNF TNF IL 15 Actin Vujanovic, L., et al. Blood (4): Butterfield, LH et al. J Immunother (3): Xu, J et al. Blood, 2007, 109 (8):
6 Major Question: Can Ad.DC recruit NK cells and how? Can Ad.DC recruit NK cells in vitro and in vivo? Which chemokines Ad.DC produce? Which chemokine receptors NK cells express? Which chemokines produced by Ad.DC effectively induce NK cell recruitment?
7 In vitro Experimental set up Day 0 Monocytes Blood donation from a healthy donor Day 5 idc AdV LacZ LPS/IFN γ Day 6 idc Ad.DC mdc Second blood donation from a healthy donor Collect cell free supernatants NK Set up 1.5 h migration assay
8 Ad.DC have the ability to recruit NK cells in vitro Percent NK Cells Migrated Media idc Ad.DC mdc Cell free supernatants n = 4 p 0.05
9 In vivo experimental set up Day 0 Monocytes Blood donation from a healthy donor Day 5 Stain cells VivoTag S 750 (PerkinElmer) idc AdV Luciferase Blood donation from the same donor 3 h incubation idc Collect DC Ad.DC NK Stain cells VivoTag XL 680 (PerkinElmer) Small animals optical imaging was performed using the IVIS optical imaging system at the time of injection (0h) and 24h post injection Subcutaneous injections in C57BL/6 albino mice (Confirmed in NOD/SCID/IL2R)
10 Enlarged image overlays of the best and average examples of NK cell migration towards Ad.DC and idc Ad.DC (best) Ad.DC (average) idc (average) Overlay + Grid 0h 20 mm 24h 20 mm Chemotaxis was quantified by measuring the distance between a DC signal focus to the apex (Top), focus (Center), and bottom edge (Bottom) of an NK cell signal. The data were standardized by calculating the percent change in the determined distance.
11 Ad.DC have the ability to recruit NK cells in vivo 60.0 Top 60.0 Center 60.0 Bottom % distance covered idc Ad.DC idc Ad.DC idc Ad.DC Top Center Bottom Apex Focus Bottom edge
12 Ad.DC and mdc induce chemotaxis of both CD56 lo CD16 + and CD56 hi CD16 NK cells Pre Migration Migrated Cells 89% 91% Ad.DC 5% 4% CD16 ECD mdc 88.02% 87.66% 4.42% 6.19% CD56 PE
13 Ad.DC secrete a number of inflammation associated chemokines pg/ml pg/ml pg/ml
14 Chemokine receptors tested on circulating NK cells by FACS Ligand Receptor CD56 lo CD16 + CD56 hi CD16 CD56 lo CD16 CCL2/MCP 1 CCR2 + CCL5/RANTES CCR3 ++ CCL4/MIP 1β, CCL2, CCL5 CCR4 + CCL3/MIP 1α, CCL4, CCL5 CCR5 + CCL19/MIP 3β CCL21/6Ckine CCR CXCL8/IL 8 CXCR CXCL9/MIG, CXCL10/IP 10 CXCR % % % +++ >50%
15 Ad.DC secrete increased amounts of CXCL8/IL 8, CXCL10/IP 10 and CCL19/MIP 3β Concentration (ng/ml) CXCL8/IL 8 (n = 36) C1 C2 C3 idc Ad.DC mdc CXCL10/IP 10 (n = 21) C1 C2 C3 idc Ad.DC mdc CCL19/MIP 3β (n = 7) C1 C2 C3 idc Ad.DC mdc p < 0.05
16 Ad.DC recruit NK cells via CXCL8/IL 8 and CXCL10/IP 10 (CXCL10) (CXCL8) (CCL2) p < 0.05
17 CXCL8/IL 8 selectively recruits CD56 lo while CXCL10/IP 10 recruits CD56 hi NK cell subsets pg/ml Purified CD16 NK Cells pg/ml
18 Conclusions Ad.DC effectively recruit NK cells in vitro and, more importantly, in vivo Ad.DC secrete a number of inflammation associated chemokines Ad.DC mediate recruitment of NK cells by CXCL8/IL 8 and CXCL10/IP 10 CXCL8/IL 8 selectively recruits CD56 lo while CXCL10/IP 10 selectively recruits CD56 hi NK cell subsets
19 Acknowledgements Lisa H. Butterfield, Ph.D. Jian Shi, M.D. Angela D. Pardee, Ph.D. Hadas P. Naveh, Ph.D. Nikola L. Vujanović, M.D., Ph.D. Andrea Šobo Vujanović Stephen H. Thorne, Ph.D. Padmavathi Sampath, Ph.D. Rachel Sikorski U. Pittsburgh Vector Core Facility Andrea Gambotto, M.D. Sheri L. Rea The UPCI Immunologic Monitoring Laboratory Cathy Brown Jennifer L. Schnelbach Sylvia E. Thomas Shrader Mary Jo Buffo Sharon Sember This study was kindly supported by: University of Pittsburgh Cancer Institute (UPCI) and the Henry L. Hillman Foundation (L.H.B. and N.L.V.) NIH 1P50CA (L.H.B.) NIH RO1 DE17150 (N.L.V.) UPCI melanoma and skin cancer SPORE career development award (L.V.).
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