JAK3 inhibitor administration in vivo in chronically SIV Infected Rhesus Macaques

JAK3 inhibitor administration in vivo in chronically SIV Infected Rhesus Macaques preferentially depletes NK Cells Ladawan Kowawisatsut 1 Kovit Pattanapanyasat 1 Aftab A. Ansari 2 1 Department of Immunology Faculty of Medicine Siriraj Hospital Mahidol University Thailand 1 Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand 2 Department of Pathology and Experimental Medicine, Emory University, USA

Why study NK-17 cells in the GALT? GALT is the one of the largest lymphocyte y storage site and also the primary site of HIV/SIV replication during acute infection NK cells are important innate cells in anti-viral immunity NK cells in Toxoplasmagondii-infected mice can produce IL-17 in the presence of IL-6, IL-23 and TGF- (The Journal of Immunology 2010,184:1776-1783) 184:1776 1783) IL-17 producing Th cells (Th17) have been reported as the important cells in protection of intestinal epithelial integrity and microbial translocation during SIV infection

JAK3 inhibitor Inhibits the common gamma chain cytokine pathways such as the IL-2, IL-7 and IL-15 signaling pathway Immunosuppressive agents that has been used in the clinic for the prevention of organ transplant rejection and autoimmune diseases Oral administration of the JAK3 inhibitor in cynomolgus monkey reduced the number of NK cells and CD8+ T cell which returned to baseline levels within 3 weeks following treatment cessation (Journal of Leukocyte Biology 2004;76:1248-1255)

Pharmacokinetics of JAK3 inhibitor Groups of two rhesus macaques were administered 1, 5, 10, or 25 mg/kg JAK3 inhibitor orally and detailed safety and PK studies performed. At a dose of 10 mg/kg/day, sufficient drug trough levels were obtained that led to the preferential depletion of NK cells Dose was tolerated well with no clinical, metabolic, hematologic, renal/liver toxicity.

Aim of the study To determine the effects of in vivo NK cell depletion on viral loads and immune responses using a JAK3 inhibitor in chronically SIV infected rhesus macaques

Protocol Blood and Rectal biopsy from 6 intravenously SIVmac251- infected Rhesus macaques JAK3 inhibitor 10 mg/kg/day g for 35 days per os pre 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 weeks Peripheral blood & colorectal biopsy collection to determine frequency of total and IL-17 NK cells Plasma monitored for drug levels PBMC analyzed for levels of phosphorylated STAT-5

Methods Mononuclear cells from blood and rectal biopsy py Stimulate with 25 ng/ml PMA and 1 mg/ml Ionomycin Add 0.5 mg/ml Brefeldin A Overnight stimulation Cell surface and Intracellular cytokine staining FITC PE PerCP-Cy5 Cy5.55 PE-Cy7 APC Alexa700 PacBlue CD8 NKG2A CD16 CD56 IL-17 CD3 Live/Dead Flow cytometry analysis by LSRII

JAK3 inhibitor had modest effects on the frequencies of CD4 and CD8 T cells in PBMC and rectal biopsies * JAK 3 inhibitor CD4 T cells JAK 3 inhibitor CD8 T cells

JAK-3 inhibitor depleted NK cells in PBMC and rectal biopsy * JAK 3 inhibitor After cessation NK cells returned in PBMC, but not in rectal biopsy

JAK3 inhibitor also effects NK-17 cells in PBMC and rectal biopsy * * JAK 3 inhibitor PBMC JAK 3 inhibitor Rectal biopsy

Viral Loads in SIVmac251 chronically infected Rhesus Macaques administered JAK3 inhibitor copies/ml JAK 3 inhibitor Transient increase in viremia during JAK-3 inhibitor admin.

Viral Loads in SIVmac251 chronically infected Rhesus Macaques administered JAK3 inhibitor copies/ml JAK 3 inhibitor Transient increase of viremia during JAK3 inhibitor admin.

Conclusion The in vivo administration of a JAK3 inhibitor to chronically SIVmac251 infected monkeys depletes both total and NK-17 cells in the blood and mucosal tissue and is associated with a transient increases in viremia denoting an important role for NK cells during chronic SIV infection.

Acknowledgement Department of Pathology and Experimental Medicine Emory University School of Medicine Professor Aftab AA A.Ansari Ann Mayne Yoshi Takahashi Dawn Little Yerkes National Primate Research Center Emory University Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University Professor Kovit Pattanapanyasat The Royal Golden Jubilee Ph.D. Program, The Thailand Research Fund Grant from NIAID, NIH to Prof. A. Ansari

* * Mean ±SD PBMC Rectum Pre CP 4.80±4.36 1.92±0.88 Week 1 0.18±0.27 2.99±4.98 Week 3 0.58±0.86 0.95±1.19 Week 6 0.34±0.07 0.25±0.62 Week 8 0.31±0.12 12 0.57±0.64 Week 12 0.31±0.28 0.35±0.70