1. Graft vs Host Disease Remains the major cause of treatment related morbidity and mortality in allogeneic HCT. Caused by donor derived alloreactive T cells. B cells may also play a critical role in GVHD pathophysiology. Chronic GVHD linked most closely with the beneficial GVT response.

2. Approaches to the Prevention of GVHD Pharmacologic – CNI/MTX – CNI/MTX vs Rapa/CNI – RAPA/MMF – Post transplant cyclophosphamide Graft source – BM, PBPC, UCB T Cell depletion – CD34 Selection – ATG, Campath Immune regulatory mechanisms – Treg – NK-T – MSCs

3. BMT CTN 0402: Phase 3 Randomized Multicenter Trial comparing Sirolimus/tacrolimus vs Tacrolimus/MTX for Acute GVHD Prophylaxis Primary Objective Compare rates of acute grade 2-4 GVHD survival after HLA- matched sibling allogneic HCT Secondary Objectives 1 year relapse-free and overall survival Incidence of grade 3-4 acute GVHD Incidence of chronic GVHD Neutrophil and platelet engraftment Infection incidence VOD incidence

4. BMT CTN 0402: Tac/Sir vs Tac/MTX for aGVHD Prophylaxis Accrual completed Oct, 2011 Age 2 – 60 yo Diagnoses: Low risk: AML,ALL CML, MDS HLA matched sibling RANDOMIZERANDOMIZE RANDOMIZERANDOMIZE Tacrolimus/ Sirolimus n= 156 Tacrolimus/ MTX n= 156 Prep regimens allowed: - TBI/VP16 or TBI/Cy HCT

5. Acute GVHD Chronic GVHD Randomized Trial Incorporating F-ATG in URD Allogeneic Transplants Finke et al. Lancet Oncology 10:855, 2009

6. Cumulative incidence of extensive chronic GVHD by treatment groups overall and by prognostic subgroups. Socié G et al. Blood 2011;117:6375-6382 ©2011 by American Society of Hematology

7. TLI/ATG Reduced Intensity Conditioning Animal studies have demonstrated that novel conditioning with TLI/ATS results in protection from GVHD Pillai et al. J Immunol 178:6241, 2007 Pillai et al. Blood 113:4458, 2009 Translation to the clinic demonstrating low acute GVHD incidence and severity and low TRM Lowsky et al. N. Engl. J Med 353:1321, 2005 Kohrt et al. Blood 114:1099, 2009

8. Host NK T cells Host T cell TLI + ATG CD1d Invariant TCR APC IL-4 Naïve donor CD4 and CD8 T cells CD4 + CD25 + donor Treg Polarized donor Th2 cell IL-4 GVHD Donor HSC HOST CELLS DONOR GRAFT + +

9. TLI and ATG Conditioning ATG 1.5 mg/kg/day Day 0 Day 180 Days -11 to -7 CSA MMF TLI 800 cGy over 10 fractions Days -4 to -1Days -11 to -7 Infection Prophylaxis HSV: if +ve acyclovir 400 mg BID CMV: blood PCR weekly EBV: blood PCR every 2 weeks PCP: Septra DS BID weekends D+42 Fungus: if prior infection or URD

10. Related DonorsUnrelated Donors 1.5% 11% Kohrt et al. Blood 2009 Incidence of Acute GVHD (II-IV)

11. CD4 + CD25 + Regulatory T Cells Major population of cells which regulate immune reactions Express transcription factor FoxP3 Deficiency or mutation of FoxP3 has autoimmune consequences in animal models and humans Cell contact-dependent suppression of alloreactive responses in mixed lymphocyte reactions (MLR) Prevent organ specific autoimmune diseases in animal models (e.g. IBD, diabetes)

12. Selection of CD4+CD25+ Regulatory T Cells (U. Perugia) Cells (x10 9 ) 1060 (540-1370) 280 (202- 390) %CD4CD25 3.0 (1.5-7.45) 92.4 (90-97.1) N° cells (x 10 6 ) 330 (221-1020) 256 (185.6-365.4) %CD4CD25 high 0.3 (0.12- 0.89) 33.6 (14.4-39.6) N° cells (x 10 6 ) 36.12 (19.98 - 84) 68.6 (20.9-143) Starting fraction Final fraction CD25 CD127 CD4 FoxP3 Gate on CD4CD25 +high Gate on CD4CD25 + Fox P3 + cells 71.9 ± 15 % Immunomagnetic Selection of CD4 + CD25 + Cells 1st step: Depletion of CD8+/CD19+cells 2ndstep: Enrichment of CD25+ cells

13. TBI CD34 + CD34 + Fludarabine T regs Tcons days Treg Haploidentical Protocol – Unversity of Perugia Cytoxan TT 8 Gy TBI in a single fraction at 16 cGy/m Thiotepa 4 mg/kg/day Cytoxan 35 mg/kg/day Fludarabine 40 mg/sqm/day D’Ianni et al. Blood 2011

14. Evaluable Patients Patients with CMV reactivation Days after transplant CMV reactivation episodes Tregs Group Control Group p<0.05

15. Outcomes – U. of Perugia Event-Free Survival 12/26 (46%) Regimen Related Toxicities: – Veno-occlusive disease (3) – Multi-organ failure (1) Acute GVHD grade III-IV (2) Serious infections (7) Relapse (AML 1) Median follow-up 18.5 months (range 16.1-27.6) D’Ianni et al. Blood 2011

16. Ex Vivo Expanded UCB T reg Brunstein C G et al. Blood 2011;117:1061-1070

17. Treatment plans Brunstein C G et al. Blood 2011;118:282-288

18. Risk of GVHD following Double UCB and Haploidentical BMT with post-transplant Cy Brunstein C G et al. Blood 2011;118:282-288

19. Treatment for Acute GVHD Corticosteroids the mainstay of therapy Many phase II studies showing a variety of agents may have biological impact Day 28 response most predictive of long term outcome

20. Etanercept (N=46) MMF (N=45) Denil (N=47) Pentostatin (N=42) Complete Response 44%73%55%62% Complete Response (Excl. prior MMF) 53%73%61%64% Treatment Failure * 24%9%26%29% BMT CTN Phase II Pick the Winner Strategy * Treatment Failure = no response, progression, or addition of another immunosuppressive agents by day 56. Alousi et al, Blood, 2009

21. Where now for treatment of Acute GVHD? Phase III MMF vs Placebo CTN study halted for futility New phase II trial with other agents? – ATG, Campath – ECP – MSCs – Rapamycin – Cytokines

22. Chronic GVHD Major source of morbidity and some mortality Immunosuppression with PSA and CNI remains the mainstay of treatment Alternative approaches – Rapamycin – ECP – Rituximab – TKIs – MSCs – Cytokines

23. Ratio of Treg:Tcon post IL-2 Koreth et al. NEJM, 2011

24. Long term administration of IL-2 for cGVHD Koreth et al. NEJM 2011

25. Future Risk Adapted Strategies Genetics of donor – recipient pairs Treatment of the donor – Statins Earlier detection – Biomarkers – Imaging