Establishing simian NK cell parameters for the study of NK-1R antagonists in vivo Jordan Orange, MD, PhD and Steven D. Douglas, MD November 16, 2005.

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Establishing simian NK cell parameters for the study of NK-1R antagonists in vivo Jordan Orange, MD, PhD and Steven D. Douglas, MD November 16, 2005

NK Cells Cytotoxicity - Perforin-mediated Antibody dependent (ADCC) - Trail/FAS-L-mediated - TNF-mediated Cytokine Production (NK1/NK2) - IFN- , TNF, IL-5, IL-13 Costimulation - CD40L (CD154) - OX40L J.S. Orange, in Up to Date, Rose, B.D. (ed) 2004 v12.1 NK K562 NK cells are lymphocytes important in immune regulation and host defense that are capable of being specifically activated or inhibited after the ligation of germline-encoded receptors.

J.S. Orange, and Z.K. Ballas 2005 Clinical Immunology v117

K562 Lytic Units 20 Per PBMC NK cell cytotoxicity

Paradigms of NK cell impairment during retroviral infection Expression returns after prolonged aviremia

NCRs in Simian NK cell populations

Simian Human

Simian Human

RDL P815 Blocking FO1 melanoma IgG  CD16  NKp30  NKp46

BT25 - Uninfected Whole blood analysis

IgG1 NKp30 CD16 NKp46 NKp BT25 - Uninfected Whole blood analysis

BT25 - Uninfected Whole blood analysis

Controversies in defining a Simian NK cell

% C16+/CD3- NK cell percentages

% of C16+/CD3- positive for NKp46 NKp46

% of C16+/CD3- positive for NKp30 NKp30

NOTE: ALL OF THESE SAMPLES ARE EXTRACTED & UNDILUTED; All samples had floaters after resuspension with EIA buffer. IPCP SP RUN #20

NOTE: ALL OF THESE SAMPLES ARE EXTRACTED & UNDILUTED; All samples had floaters after resuspension with EIA buffer. IPCP SP RUN #20

IPCP SP RESULTS 1 Samples BV01 and CA84 were combined because we were not sent enough sample to assay individually

IPCP SP RESULTS 2 Samples CE48 and DD95 were combined because we were not sent enough sample to assay individually 3 Original run done on EDTA plasma; re-run done on HEP plasma

IPCP SP RESULTS 4 SP run results were invalid – need to be repeated * Bloods were shipped by Tulane on ice packs; they were cold upon arrival at CHOP

SP NK-1R NK cell Activating receptor Cytotoxicity Cytokine production Activating receptor expression Inhibitory receptor expression Ca ++ flux Chemotaxis Aim 3a SP inhib. Signal-mediated SPA NK-1R signaling Activation signaling

NK cell SP NK-1R Cytotoxicity Activating receptor expression 2 1 On treatment Pre-treatment Post-infection in treatment evaluation Aim 3b HIV infected cell SPA