Induced Pluripotent Stem Cells and Human Primary Immunodeficiencies Luigi D. Notarangelo Division of Immunology Children’s Hospital Boston [email protected] Studying PID pathophysiology: Limitations of current approaches - Limited availability of biological samples - Differences in immune system (rare diseases, mostly affecting children) development and function vs. humans -Genetic and phenotypic heterogeneity - Limited diversity of mutations (limited information of impact at the and of phenotypic heterogeneity cellular level) - Inbred strains limit analysis of -Studies mostly restricted to blood cells, modifying genes but little is known about pathophysiology of extraimmune manifestations - Relevance of humanized mouse models • developmental defects: DiGeorge, CHH is mostly limited to hematopoietic and • defects of DNA repair lymphoid phenotype, and is problematic • tissue-specific disease (HSE) - Elevated costs The Potential of iPSC Research Tissue development Disease models Gene correction/repair • Skin fibroblasts Drug screening • Blood cells • Keratinocytes cell therapy ALS Alzheimer’s Cancer PIDs CF Diabetes IBD OCT4 cMYC KLF4 SOX2 Transduction/ Stem cell Transfection biology iPSCs Patient-derived iPSCs for disease modeling and drug discovery in PID Reticular dysgenesis • AR SCID with extreme lymphopenia and agranulocytosis • Myeloid differentiation in the bone marrow blocked at promyelocyte stage • Sensorineural deafness • Fatal, unless treated by HCT • Mutations of adenylate kinase 2 (AK2) gene affect balance of ADP, disrupt mitochondrial membrane potential and cause apoptosis Modeling human SCID with induced pluripotent stem cells (iPSCs) +! sortsort CD34 CD43++ CD43 CD43–-! ! 9-11 days! hemogenic progenitors! ! + cytokines,! growth factors ! hiPSC EB! CD34 CD43! OP9-DL4! Coculture! FACS Analysis! ,! ")'! ")'! %&*! %&'(! %&('! %&*! %! ")'! "#)! "#$! %&'! %&$! %&(! d21 d28 d35 d42 d42 T-B-NK+ SCID (R394W; R394W) Omenn syndrome (K86fs; E722K) Control Control SCID Omenn Clonotypic diversity Vβ usage Percentage (%) Percentage (%) Percentage (%) TCRVβ TCRVβ TCRVβ CDR3 length Percentage (%) Percentage (%) Percentage (%) NucleoBdes NucleoBdes NucleoBdes HerpesHerpes simplex encephalitis encephalitis " " 50" 100" 10% consanguinity 40" 80" HSE" 30" HSV+" 60" 20" 40" 14% consanguinity! 10" 20" Numberof cases HSE with 0" 0" Seroprevalenceof HSV+(%) 0" 10" 20" 30" 40" 50" 60" 70" 80" Age (years) " • Herpes simplex encephalitis (HSE) is a very rare but severe complication of infection due to HSV-1, a ubiquitous and otherwise inoffensive virus that is transported to the CNS through olfactory and trigeminal nerves. • HSE is the most common cause of sporadic viral encephalitis in Western countries, occurring at a rate of 1-2 per 500,000 individuals per year. • Use of acyclovir has improved survival (overall mortality rate 2.5%), but 35-62% of affected individuals have significant neurologic sequelae. HSE: a diverse collection of inborn errors of TLR3-IFN-dependent immunity HSV-1 TLR3 TRIF IL10RB UNC-93B IFN-λR NEMO TBK1 TRAF3 TYK-2 IKK-ε IKK complex STAT-1 IFN-α/βR IRF9 JAK-1 IRF-3 NF-κB STAT-2 IFN-λ IFN-β IFN-α Differential IFN response in fibroblasts and PBMC from patients with defects of TLR3 pathway !" !" !" #$"%&$'" #$"%&$'" #$"%&$'" #("%&$'" #("%&$'" #("%&$'" #$")*!+'," 3456" #$")*!+'," 3456" 3456" #$")*!+'," 12 12 12 "0 "0 "0 ! ! ! -.*/ -.*/ -.*/ .@C=EC5<DFD" "0-)3456" "0-)3456" "0-)3456" " " " -.*/ -.*/ -.*/ *7""""""""""""""""878""""""""""""""""""""""""""""""""*7""""""""""""""""978/:"""""""""""""""""""""""""""""""*7""""""""";<=<---"""""""">?!8"""""""7@ABC@D" !" #$"%&$'" ;,?!" !"#$%&'%()*+%,%-./%0&1%23445% Hypothesis HSE in patients with impaired TLR3 signaling results from defective production of IFN-α/β or –λ by CNS-specific cell types Questions Which CNS cell type(s) critically require intact TLR3 signaling for anti-HSV-1 immunity? Which IFN is involved in mediating protection? Impaired induction of IFNs after HSV-1 infection in UNC93B-deficient neurons and oligodendrocytes Neurons! Oligodendrocytes! ! ! 12! * * 250! C+ (hESC)! * C+ (hESC)! * * C+ (iPSC)! 10! C+ (iPSC)! 200! !"!# !"#$%&'!"!# !"#$%&'UNC-93B-/-! 8! UNC-93B-/-! 150! 6! 100! 4! mRNA fold induction fold mRNA 1 mRNA fold induction fold 1 mRNA " 50! 2! ! IFN- 0! IFN- 0! NS! HSV-1! NS! HSV-1! ! Astrocytes! ! NSCs! 160! C+ (hESC)! 300! C+ hESC! C+ (iPSC)! C+ (iPSC)! 140! 250! !"!# UNC-93B-/-!"#$%&'!"!#! 120! !"#$%&'UNC-93B-/- ! 200! 100! 150! 80! 60! 100! 1 mRNA fold induction fold 1mRNA ! 40! 1 mRNA fold induction fold 1mRNA 50! ! 20! IFN- 0! IFN- 0! NS! HSV-1! NS! HSV-1! (Lafaille et al., Nature 2012) TLR3- and UNC-93B-deficient neurons and oligodendrocytes are highly susceptible to HSV-1 replication A Neurons Oligodendrocytes NSCs Astrocytes B Neurons (Lafaille et al., Nature 2012) Gene transfer and IFN-α rescue susceptibility of TLR3- and UNC-93B-deficient neurons to HSV-1 replication 12000 HSV-1-GFP 10000 HSV-1-GFP + IFN-α2b 8000 6000 4000 2000 HSV-1-GFP measurement 0 Control UNC93B1-/- UNC93B1-/- TLR3-/- TLR3-/- C+ + Mock UNC-93B-/- + UNC-93B-/- + TLR3-/- + Mock TLR3-/- + TLR3 + mock + mock + UNC93B1 + mock + TLR3 Mock UNC93B1 (Lafaille et al., Nature 2012) • Neurons and oligodendrocytes provide intrinsic protective anti-HSV-1 immunity in the CNS, through a TLR3-, IFN-α/β- Intrinsic immunity (type 1 IFN) Extrinsic protecBon (type 1 IFN) dependent pathway • Patient-derived iPSCs represent a powerful platform for modeling extra-hematopoietic manifestations of PIDs. • Need to define: Extrinsic protecBon (type 1 IFN) - mechanisms of protection (basal IFN-1 production?) - effect on virus latency - impact on other neurotropic viruses - effects on microglia Acknowledgements Division of Immunology, CHB MSKCC Itai M. Pessach Lorenz Studer Jose Ordovas-Montanes Fabien G Lafaille Kerstin Felgentreff Katja Weinacht Division of Hematology, CHB Stefano Volpi Thorsten Schlaeger Kerry Dobbs Boston University Gustavo Mostloslavsky Rockefeller University Jean-Laurent Casanova Sunnybrook Research Institute Shen-Ying Zhang Juan Carlos Zúñiga-Pflücker Funding NIH 1R03AI0883502-01 NIH 1R21AI089810-01 NIH 1R01NS072381-02 NIH 1R01AI100887-01 .