A TOLLIP DEFICIENCY ALLELE, RS , IS ASSOCIATED WITH LNCRNA TOLLIP-AS1 EXPRESSION, T-CELL MEMORY PHENOTYPE, AND INCREASED TB SUSCEPTIBILITY

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1 A TOLLIP DEFICIENCY ALLELE, RS , IS ASSOCIATED WITH LNCRNA TOLLIP-AS EXPRESSION, T-CELL MEMORY PHENOTYPE, AND INCREASED TB SUSCEPTIBILITY February 2, 208 5th Global Forum on TB Vaccines

2 MULTIPLE FACTORS INFLUENCE VACCINE IMMUNITY AND TB SUSCEPTIBILITY Pathogen Exposure Host Genetics Age Profile Genes Associated with Mycobacterial Susceptibility Determine Pathways Critical to Mycobacterial Control Improve Therapies and Vaccines Case courtesy of Dr. Dalia Ibrahim, Radiopaedia.org, rid: 30824

3 BCG Activity TNF; IL-6 BCG TOLLIP TLR, 2, 4, 6 IL-R IL-2 IL-23 IL- TB control/ progression T cell

4 TOLLIP CRITICAL REGULATOR OF IMMUNE ACTIVATION Chr (-) rs rs ESCRT rs rs rs rs rs rs rs TBD C2 CUE Autophagy Endosomal Localizatio n Ub binding TLR2, TLR4, IL-R binding MyD88 TOLLIP IL-R IRAK cytokines TLR2,TLR 4 IKKs I-kB TF activation

5 TNF (pg/ml) [Nitrite] (µm) IL-6, pg/ml rs TNF (pg/ml) F (pg/ml) TOLLIP DEFICIENCY INDUCES INFLAMMATION G *** itrite] (µm) TOLLIP mrna / GAPDH rs rs C G A A. CRISPR/Cas9 knockout of TOLLIP in THPs Empty vector control (EV) vs TOLLIP-KO B Control TOLLIP- KO *** *** EV KO a-tollip G RLU EV KO A Control TOLLIP-KO b-actin none LPS Control TOLLIP-KO 00 IL-6 (pg/ml)000 0 RLU *** *** PAM2 ** LPS ng/ml ** C/C C/G G/G Seattle Healthy RLU Control Cohort IL-6 (pg/ml)0000 *** C/C G/C G/G0 PAM3 250ng/ml *** C/C C/G G/G none Con TOL 0 Media LPS MTb none Control TOLL IP- KO * * MTb 5 Shah et al J. Immunol Control TOLLIP-KO *

6 rs rs TOLLIP mrna / GAPDH rs RS IS A FUNCTIONAL TOLLIP PROMOTER VARIANT A G South African cohort Infant Monocytes Community Control N = 55 TOLLIP -362 Luciferase RLU TOLLIP -362 Luciferase HEK293T cells TOLLIP promoter transfected with Luciferase protein Luminescence measured after 24hr G A G C RLU * 0 0. C/C G/C G/G * p = 0.008, Kruskal-Wallis test RLU Shah et al (207) AJRCCM 96:502-5

7 TOLLIP mrna TOLLIP mrna TOLLIP mrna RS INFLUENCES TOLLIP-AS EXPRESSION AND TOLLIP MRNA TRANSCRIPTION TOLLIP- AS TF TOLLIP TOLLIP-AS mrna control AS sirna AS sirna2 24hr 48hr 72hr control AS sirna AS sirna2 24hr 48hr 72hr rs TOLLIP-AS mrna TOLLIP-AS mrna

8 HYPOTHESIS: ONE OR MORE FUNCTIONAL TOLLIP VARIANTS REGULATE IN VIVO IMMUNE RESPONSES TO BCG & SUSCEPTIBILITY TO TB Collaborators: University of Cape Town Willem Hanekom Mark Hatherill Tom Scriba Munya Musvosvi Muki Shey

9 Evaluating BCG-specific T cell Immunity In TOLLIP-deficient infants BCG N = 74 Infant Whole Blood BCG-specific T Cell Cytokines IL-2 TNF IFN-g IL-7 IL-22 IL-4 FSC-H CD3 FSC-A CD8 CD4 CD4 CD8 SSC-A FSC-A IL-2 Media TNF Shah et al (207) AJRCCM 96:502-5

10 Stimulation Index TOLLIP DEFICIENCY REDUCES BCG-SPECIFIC IL-2 AND PROLIFERATION IN CD4 T CELLS % CD4 +, IL * * C/C G/C G/G 0.0 C/C G/C G/G C/C G/C G/G * P = 0.02 * P = 0.04 Shah et al (207) AJRCCM 96:502-5

11 MULTIPLE FACTORS INFLUENCE MEMORY T CELL PHENOTYPES Inflammation Ag Exposure Proliferation/ Duration IL-2 TNF IFNg Apoptosis Naïve Tcm Tem Teff Texh Dead

12 TCM Naive TEM CCR7 TEMRA CCR7 CD45RA

13 High Low TOLLIP Naïve TCM TEM TEMRA IL-2+ TNF+ *p = 0.02, partial permutation test IL-2+ TNF+

14 % of CD4+ T cells TOLLIP-DEFICIENT INFANTS DEMONSTRATE FEWER TCM OVERALL AT WEEKS OF AGE 0 * NAIVE TCM TEMRA TEM NAIVE TCM TEMRA TEM C/C or G/C G/G

15 TOLLIP DEFICIENCY IS ASSOCIATED WITH ADULT TB SUSCEPTIBILITY N Cord Blood Controls (N) 762 Adults with Pulmonary TB (N) 385 Adults with Meningeal TB (N) 307 HIV-infected (%) 0 Age (Mean) 38 Sex (% female) 37 Analysis SNP Group P OR rs control (recessive) TB all 6.64E-07.7 Jeremy Farrar Sarah Dunstan The Hospital for Tropical Diseases Pham Ngoc Thach Hospital for Tuberculosis Oxford University Clinical Research Unit Shah et al J. Immunol 202

16 TOLLIP Deficiency is Associated with Latent Tuberculosis Susceptibility N No LTBI (N) 6 Adults with LTBI (N) 50 HIV-infected (%) 0 Age (Mean) 42 Sex (% female) 46 Analysis SNP Group P (geno) OR rs No LTBI (recessive) LTBI 7.0E (. 7.) David Horne; Masa Narita King County Public Health Department Harborview Medical Center Seattle, WA Shah et al AJRCCM 207.

17 TOLLIP deficiency BCG TNF; IL-6 TOLLIP TLR, 2, 4, 6 T cell differentiation Altered memory response CD4+ T cell Reduced vaccine duration? Increased TB pathology?

18 SUMMARY A functional single nucleotide polymorphism alters TOLLIP promoter activity in monocytes Antisense RNA TOLLIP-AS influences TOLLIP expression (and perhaps innate immune responses) TOLLIP deficiency is associated with altered T cell phenotypes from a very early age TOLLIP variation is associated with TB in multiple genetic case control cohorts

19 ACKNOWLEDGEMENTS CERID Melanie Brown Tom Hawn Rick Wells Glenna Peterson Monica Campo Andrew Graustein UC Berkeley Jeff Cox King County Public Health David Horne Masa Narita University of Cape Town Tom Scriba Mark Hatherill Willem Hanekom Munya Musvosvi Oxford University Clinical Research Unit Sarah Dunstan Guy Thwaites UW Dept of Immunology Dan Stetson Betsy Gray Kenya Grace John-Stewart AJ Warr Uganda Cathy Stein Henry Boom NIH, VA R&D UW School of Medicine Bill and Melinda Gates Foundation