1. Probiotic and IL-21 Treatment Promotes Th17 Cell Recovery in ARV-Treatment of Pigtail Macaques Alexandra M. Ortiz Laboratory of Jason Brenchley July 23, 2014 Abstract #: WEAA0104

2. Th17 Cells and Probiotics in HIV-associated Intestinal Immune Dysfunction HIV is characterized by persistent GI immune dysfunction. Preferential Th17 loss Th17 are antimicrobial CD4+ T-cells essential for intestinal immunity. Generated in the context of commensal signaling Immune dysfunction correlates with microbial translocation and dysbiosis Probiotic supplementation of chronically-infected, ARV-treated macaques: Increased colonic CD4+ T-cell function and frequency Increased function but not frequency of colonic Th17 cells IL21 therapy in acutely SIV-infected macaques promotes Th17 retention during treatment (Pallikkuth PLoSPathog 2013) (Klatt JCI 2013)

3. Experimental Design Animals: 11 Pigtail Macaques (Maccaca nemestrina) 6 animals treated with ARV + Probiotics (Pb) + IL-21, 5 ARV-only controls Infection: 3000 TCID50, SIV mac239 i.v. ARVs: 20-30 mg/kg PMPA i.v., 30mg/kg FTC i.v., 240 mg L’812 – daily Probiotics: 10 11 live bacteria (VSL#3) oral - daily Bifidobacterium: breve, longum and infantis Lactobacillus: acidophilus, plantarum, paracasei, bulgaricus Streptococcus thermophilus IL-21: 50 ug/kg IL-21, s.c. – 5 weekly doses, early and late chronic RM IL-21 fused to RM IgG2 Fc, produced in S-2 cells

4. IL-21 and Probiotic Therapy do not Interfere with Viral Suppression Viral Load (log copies/mL) Day (relative to infection) PTA2P031  Treatment initiation (late) IL21 (early) IL21

5. IL-21and Probiotic Therapy Improve Intestinal CD4+ T-Cell Frequency Jejunum Rectal Biopsy Day (relative to infection) Percent CD4+ of CD3+ T-cells ARV only ARV+Pb+IL21 PTA2P031

6. IL-21and Probiotic Therapy do not Induce CD4+ T-cell Activation PBMCLN BAL JejRB Day (relative to infection) Percent Ki67+ of CD4+ TM ARV only ARV+Pb+IL21

7. IL-21and Probiotic Therapy Improve Intestinal Th17 Frequency Percent IL17+ of memory CD4+T-Cells Day (relative to infection) Jejunum Rectal Biopsy ARV only ARV+Pb+IL21

8. IL-21and Probiotic Therapy Improve Polyfunctional Th17 Recovery Percent Polyfunctional of CD3+ T-Cells Day (relative to infection) Jejunum Rectal Biopsy ARV only ARV+Pb+IL21

9. IL-21 and Probiotic Therapy Moderately Reduced Colonic IDO Expression ARV only ARV+Pb+IL21 Percent IDO+ Tissue Colon Mesenteric LN

10. IL-21and Probiotic Therapy Reduce Comorbidities Percent Incidence-Free Animals Day (relative to infection)

11. Conclusions and Future Directions Therapeutic treatment with probiotics and IL21 improved disease progression in ARV-treated macaques as measured by: – Intestinal CD4+ T-cell recovery – Improved intestinal Th17 frequency and functionality – Decreased incidence of health complications By what mechanism are Th17 cells expanded? Would treatment promote enhanced protection against microbial challenge? Does probiotic and IL21 treatment reduce microbial translocation and dysbiosis?

12. Acknowledgements Brenchley Lab Jason Brenchley Nina Calantone Kirby Carmack Sarah DiNapoli Zachary Klase Molly Perkins Carol Vinton Veterinary Staff Heather Cronise Richard Herbert Joanna Swerczek LMM Core NCI-NIH Jake Estes Emory University – IL21 Mirko Paiardini Francois Villinger Gilead – PMPA/FTC Michael Miller Merck – L’812 Daria Hazuda Sigma-Tau – VSL#3 Claudio De Simone