1. Severe Combined Immunodeficiency Syndrome A heterogeneous group of congenital disorders which results in the absence of antigen-specific T and B cell responses  1/50-100, 000 live births  Newborn screening estimate now 1/30-40,000 live birth  Athabascan-speaking Native Americans, an incidence of 52/100,000 live births Early classification based on presence or absence of adenosine deaminase and the % and absolute numbers of T, B, and NK cells

2. Genetic mutations associated with SCID GeneFunctionPhenotypeChromosomal location Year Published ADA Purine salvage enzyme Recycles adenosine and deoxyadenosine after DNA breakdown T, B-, NK+20q12-13 1972 TCR abn Signaling through TCR T lo, B+,NK+11q23 1987  chain Common g chain (IL-2,4,6,15,21) T-,B+, NK-Xq13 1993 ZAP 70 Tyrosine Kinase CD8 def2q12 1994 Janus kinase- 3Tyrosine kinase Signaling through  C T-, B+, NK-19p13 1995 RAG1, RAG-2 Omenn's syndrome Recombinase activating genes initiation of VDJ recombination T-B-NK+ T+, B-,NK+ 11p13 1996 IL7-Ra Cytokine Receptor T-, B+, NK+5p13 1998 ArtemisDNA repair enzyme T-B-NK+10p 1998

4. Diagnosis and Outcome of infants identified by newborn Screening DiagnosisEvaluationTreatmentOutcome #1ADA deficiency now 17 months old 34 lymphs/ulPEG ADANormal T cell function Vaccinated Successfully #2X linked + FH Now 16 months B+, NK+, T-TCD HLA-MM Related BMT Normal T cell function #3ADA deficiency Now 16 months old 117 lymphs/ul 85% NK, 15% B PEG ADANormal T cell function Vaccinated Successfully #4NormalNL phenotypeNL T cell funcNL B cell function #5Transient T lymphopenia Now 9.5 months old CD3-233/ulT cell numbers Normalized NL T cell functionVaccinated Successfully IgG3 deficient #6T lymphopenia NL ADA, IL-7R, nl NL CD3  Now almost 9m CD3:741/ul, inc B CD4:613/ul CD8:117/ul T cell numbers Increasing but not normal NL T cell functionVaccinated Successfully #7X linked Now 4 months old B+, NK+, T-TCD HLA –MM RelBMT 3+ m, well No GVHD T cells coming in #8Likely X-linked NL ADA, PNP 3 weeks old ALC 1450, B 1450/ul No T, NK 29/ul Being HLA typedPre HCT

5. Laboratory Evaluation  Lymphoid phenotype  PHA, MLC, NK function  ADA and PNP levels  Immunoglobulin levels  HIV Ab parents, HIV PCR child  HLA Class I and Class II expression  +/- phenotyping for CD127 (IL-7R)or CD132 (IL-2  chain) if consistent phenotype

6. Response to PEG-ADA 20% show no response Majority see T cell immune response in the short term but it wanes with time B cell immunity, 50% will need continued IVIG –B cell numbers increase within a few weeks of therapy –T cell numbers may require several months to increase

7. RESPONSE TO PEG-ADA in two infants identified by NBS

8. TetDiphPertHIB Pre0.811.016 Post3.431.39403 PRE 1(0.6)3(1.40)4(0.0)14(3.4)19(2.0)23(0.9)6B(1.20) 7F(1.3)18c(0.90) POST PCV13x3 1(8.1)3(>37.6)4(6.5)14(2.1)19(7.4)23(3.3)6B(11.8)7F(33.9)18C(12.3 ) TetDiphPertHIB Pre1.210.651.61 Post6.79>2.537>9 PRE 1(0.8)3(>37.5)4(0.5)14(2.1)19(1.2)23(0.6)6B(0.7)7F(4.4)18c(0.6) POST PCV13x3 1(9.3)3(34.4)4(4)14(1.6)19(6.4)23(1.3)6B(3.9)7F(18.1)18c(6.6) Response to PEG-ADA

9. Results of HCT for ADA deficiency Publication HLA MSIBAlternative donor Eur Experience* 81%, n=1929%, n=26 MSKCCnone50%, n=8 Ulm, Munich100%, n=763%, n=8 81-100% 29%-63% *50% survivors had neurologic deficits

10. Newborn Screening  Allows early identification and treatment of affected infants  EFS if HCT <2 months of age: 90-100%  Currently how well young infants tolerate cytoreduction is unknown  How much cytoreduction to ensure B cell engraftment unknown  NBS also uncovers infants who may have transient lymphopenia or immune defects that previously were unrecognized and may or may be associated with an increased risk of infection.  Need to determine best way to inform parents  Current literature needs to be translated into multiple languages  Available literature for parents is either too simplistic or too complicated

11. Normal TRECS No TRECS Longitudinal lymphoid phenotype of female child with low-absent TRECS on newborn screen

12. Lymphoid phenotype and function AgeALCCD3CD4CD8CD19CD56 %CD4/ CD45RA PHAOKT3 2 months175022814053735770 160,143 13, 478 2.5 months196655035417712192160.33189, 056 3 months267485650832114445080.47 3.9 months2700116167545912693510.72 5. 1 months40902536159490012682860.79

13. Age at test PHA LLN 109, 576 OKT3 LLN 3, 715 Candida LLN: 6624 Tetanus Toxoid LLN:2417 Vaccine Responses 1.9 m 2628811010323283 2.6m 14405077453 4.2m 100222 1050 5.7m 185539317494673 6614 + Response to Tetanus, Pertussis, Diphtheria PCV13, HIB Longitudinal immune phenotype and function of female infant with low but detectable TRECs on newborn screen

14. Why do children with SCID ever need cytoreduction Effect of pre-HCT NK function on engraftment following TCD MM- related SBA- E- BMT in the absence of cytoreduction  To ensure donor B cell engraftment  To prevent graft rejection in patients with NK activity via KIR pathways  In pts with ADA deficiency-to ensure 100% donor chimerism including red cell lineage  To prevent T cell mediated graft rejection in those with PHA >5% LLN PRE HCT NK function EngraftedFailed to engraft Absent 14/140/14 Present1/8 7/8 CHI-SQUARE=17.9, p <.001

15. Results of unmodified HLA-matched related BMT Matched sibling transplant remains the treatment of choice Cytoreduction or GVHD prophylaxis generally unnecessary European Experience 1968-1999: 81%, n=104, –ADA def: 81%, Reticular Dysgenesis: 75%- HLA matched BMT Single Center –MSKCC: 1971-2011, 87.5% survival, n=16 –Hopital Necker-Enfants Malades 1971-92, 80% survival, n=30 –Univ of Ulm: 81% survival, n=21 –DUMC: 1982-98, 100% survival, n=12 –Univ of Brescia: >80%, n=35

16. T and B cell function following unmodified HLA- matched BMT for SCID  Normal T cell numbers rapidly restored 2-4 weeks  Normal T cell mitogen and specific antigen responses restored  B cell function returns by one year post transplant

17. What about patients who lack HLA matched sibling donor TCD HLA Mismatched BMT  Depending on efficiency of T cell depletion  Current outcomes: 70-80%, and if HCT <2 months: 90-100%  Low incidence of GVHD  Parent readily available

18. Probability of Overall Survival after Unrelated Donor Transplants for SCID, 1990-2004, n=200 Probability Years Overall survival, 60% (95% CI 52-67) @ 5 years  No significant advantage over parental T cell depleted HLA non-identical marrow grafts when comparable preparative regimens are used  Potential risk of infectious complications due to time needed to procure a donor  Increased Risk of GVHD

19. Probability of Overall Survival after Unrelated Donor Transplants for SCID, 1990-2004, n=200 Probability Years Overall survival, 60% (95% CI 52-67) @ 5 years  No significant advantage over parental T cell depleted HLA non-identical marrow grafts when comparable preparative regimens are used  Potential risk of infectious complications due to time needed to procure a donor  Increased Risk of GVHD

20. Probability of Overall Survival after Unrelated Donor Cord Blood Transplants for SCID, 1990- 2005, n=76, CIBMTR Probability of survival Years Post HCT Overall survival, 57% (95% CI 44-69) @ 5 years CIBMTR data, 2007  Immediately available unlike MUD HCT  Viral naïve, CMV seronegative  Less acute and chronic GVHD despite 1-2 Ag MM  No clear advantage over HLA MM-Rel HCT  Higher risk of GHVD, appox 25% aGVHD, 13% chronic

21. Immune Function 50% of children have normal T cell function in the first 6 months post BMT 80% have normal T cell function by 6 to 12 months post BMT However, without cytoreduction, lack of donor B cell engraftment, IVIG dependent

22. Recovery of T cells post SBA- E- parental BMT for SCID Months post Mis-related parental TCD BMT for SCID Median CD3+ cell count

23. Recovery of CD4+ T cells post SBA- E- parental BMT for SCID Months post Mis-related parental TCD BMT for SCID Median CD4+ cell count

24. Numbers of circulating naive CD4+ T cells post HCT For SCID

25. Comparison of the percentage of children with normal PHA post SBA- E- BMT for SCID: With and Without pre- transplant cytoreduction

26. Median serum IgG Levels post Mis-matched TCD BMT for SCID

27. Median serum IgA Levels post Mis-matched TCD BMT for SCID

28. Conclusions and Future directions  The majority of children with SCID can be cured by a HCT providing it is performed early  Primary immunodeficiency Treatment Consortium (PIDTC)  Retrospective study to determine variables associated with outcome  Prospective study to determine outcome of patients identified through newborn screening  HCT  Gene therapy  PEG-ADA  Trial under development to determine lowest amt of cytoreduction to ensure T and B cell engraftment  Need to determine the best way to inform parents of NBS which require further evaluation  Need to develop ways to promptly direct parents to centers which can quickly evaluate and treat children with PID