Modulating STAT Signaling to Promote Engraftment of Allogeneic Bone Marrow Transplant Jacopo Mariotti, M.D. Center for Cancer Research, NCI, NIH Istituto Nazionale Tumori, Milano Verona; May 22, 2009
Non-myeloablative vs Myeloablative HSCT Risk of Rejection Treatment Related Mortality (Giralt, Blood 1997) Risk of Graft Rejection for HLA-matched related transplants (McSweeney, Blood 2001) Immunosuppressive Treatment is necessary for achieving engraftment with non-myeloablative HCT Risk of Graft Rejection especially for related HLA-mismatched or MUD transplants and/or after T cell depletion (Maris, Blood 2003; O Donnell, BT 2002) Research Goal: Identify new strategies (immunomodulatory agents, functionally-defined T cell populations) that allow engraftment without significant GVHD and reduced immunosuppression
Role of STAT Signaling in Immunologic Response Laurence, Nat Immunol 2007
Role of STAT Signaling in allogeneic HSCT Graft Versus Host Disease Donor STAT4 -/- T cells ( Th1) Donor STAT6 -/- T cells ( Th2) Inhibition of STAT1 ( Th1) Graft Rejection Host Th1/Tc1 T cells Host Th2/Tc2 T cells Host STAT1 -/- T cells ( Th1) GVHD mortality (skin) GVHD mortality (GI, weight loss) Nikolic, JCI 2000 GVHD mortality Mapara, Exp Hematol 2006 Graft rejection Graft rejection Graft rejection Mariotti, Blood 2008
Graft rejection Model: TBI and Host T Cell Add-back (Reisner, Blood 2003; Mariotti, JI 2009) 10.05 Gy TBI (BALB/c) 11 Gy TBI (B6) Purified Host T cells add-back (0.1x10^6) TCD (10x10^6) ± Th2R Day -2 Day -1 Day 0 Spleen Harvest Syngeneic DC Stim (HVG) Allogeneic DC Stim (GVH) Day 5-14 Day 90 Overall Survival Weight Change Long-term Engraftment Detection of Allospecific IFN-γ secretion Characterization of Cytokine Secretion (Th1 vs Th2)
Th1/Tc1 >> Th2/Tc2 for mediating Graft Rejection vs. P<0.0005, vs. P<0.0005 B6 BALB/c 100 100 Donor alone Host T Th1/Tc1 Th2/Tc2 Survival (%) 75 50 25 0 0 15 30 45 60 75 90 Time after T (d) Percent Donor 75 50 25 0 alone + Th2/Tc2
STAT1 signaling is Indispensable for Graft Rejection BALB/c B6 100 vs. P<.01 Donor only Host Stat1 -/- T WT T Survival (%) 75 50 25 Chimerism-d90 0 0 25 50 75 100 Time after T (d) 100 Percent Donor 75 50 25 0 Spleen Blood Spleen Blood only + Stat1 -/- T
Prevention of Graft rejection: role of STAT4 and STAT6 STAT4: STAT4-/- ( Th1) host T cells Graft Rejection IL-12 Infusion host Th1 Graft Rejection STAT6: STAT6-/- ( Th2) host T cells Graft Rejection WT host T cells + donor Th2R Graft Rejection STAT6-/- ( Th2) host T cells Graft Rejection with donor Th2R CD8 + INFγ + splenocytes(10 6 ) 3.5 3.0 2.5 2.0 1.5 1.0 0.5 * * * * * * * * * * * * Syngeneic DC stimulation Allogeneic DC stimulation *** p<.001 Host inocula Donor inocula 0 _ T _ T STAT6 -/- T WT T /Th2R STAT6 -/- T /Th2R IL-4 -/- T /Th2R
Objectives of the Project Aim 1: Elucidate the kinetics of STAT activation and cross-talk in host T cells during graft rejection and tolerance Aim 2: Determine the cross-inhibition between different STAT molecules that dictate Th1- or Th2-type polarization of host T cells during graft rejection and tolerance Aim 3: Develop new strategies of blocking host Th1-type polarization in order to prevent graft rejection and reduce pre-transplant conditioning and post-transplant immunosuppressive treatment
Aim1: Validation In Vitro of the Methods Host Donor C57/BL6 C57/BL6 BALB/c BALB.B Spleen Harvest Mixed Lymphocyte Reaction (MLR) Different Host:Donor T Cell Ratio STAT signaling expression of STAT1/3/4/5/6 phosphorilation status of STAT1/3/4/5/6 Phospho Flow-Cytometry (Nolan, JI 2005) Methods Real Time PCR Western Blotting Validation of flow-cytometry findings
11 Gy TBI Aim1: In Vivo Analysis of STAT Signaling Lethal Body Radiation and Host T Cell Add-back Advantage: able to quantify the Host Immunologic Barrier for engraftment Purified Host T cells add-back (0.1x10^6) TCD (10x10^6) ± Donor Th2 Cells Day -2 Day -1 Day 0 Day 1-3-5-8-11-14 Progressive Reduction of TBI Dose Advantage: able to consider Thymus reconstitution as a variable Characterization of Host T Cells and DC STAT Signaling by Flow-cytometry From 11 Gy to 2 Gy TBI TCD (10x10^6) ± Donor Th2 Cells Day -2 Day 0
Aim1: Role of STAT3 and STAT5 11 Gy TBI (B6) Purified Host T cells add-back STAT3 fl/fl Cre STAT5 fl/fl Cre TCD (10x10^6) Day -2 Day -1 Day 0 Spleen Harvest Day 7 Day 90 Detection of Allospecific IFN-γ secretion Characterization of Cytokine Secretion (Th1 vs Th2) Overall Survival Long-term Engraftment STAT3 fl/fl Cre and STAT5 fl/fl Cre will be provided from J. O Shea laboratory
Aim2: Cross Inhibition between STAT molecules Hypothesis: STAT6 and STAT1 cross-inhibition controls Th2 and Th1 differentiation In vivo 11 Gy TBI (B6) Purified Host T cells add-back STAT6-ER TCD (10x10^6) + 4-hydroxy-tamoxifen Day -2 Day -1 Day 0 Analysis of host T cells expression of STAT1 and STAT4 at different doses of 4-hydroxy-tamoxifen STAT6-ER: STAT6-estrogen receptor fusion protein (Kurata, Immunity 1999)
Aim2: Cross Inhibition between STAT molecules In vitro (MLR) Hypothesis 1: STAT6 dimerize with STAT1 when there is an excess of STAT6 signaling STAT6 Jak1 Tyk2 1 1 Method Isolate host T cells under condition of graft rejection (w/o Th2 cells) or tolerance (w Th2 cells). Immunoprecipitation of STAT1 and STAT6
Aim2: Cross Inhibition between STAT molecules In vitro (MLR) Hypothesis 2: STAT6 is erroneously recruited on the receptors binding sites of IFN-I and -II or IL-12 and inhibits the binding of STAT1 Method Isolate host T cells under condition of graft rejection (w/o Th2 cells) or tolerance (w Th2 cells). Immunoprecipitation of STAT6 and Type -I or -II IFN or IL-12 Receptors. Henninghausen, Gen & Dev, 2008
Aim3: New Strategies for Non-Myeloablative T Background Inhibition of STAT1 Histone Deacetylase Inhibitor (i.e. SAHA) abrogates STAT1 and STAT3 signaling and reduces GVHD (Mapara, 2006) Vorinostat has a proven anti-tumor activity(martinez-iglesias, 2008) Inhibition of JAK signaling CP-690,550 is a highly selective JAK3 inhibitor, with no effect outside the immune-system (similarly to JAK3-SCID patients) On the contrary calcineurin inhibitors, mtor inhibitors, steroids have systemic toxicity CP-690,550 prevents heart and kidney rejection(non-human models) CP-690,550 is effective in clinical trials for psoriasis, RA Approval from FDA is expected within 2009.
Aim3: New Strategies for Non-Myeloablative T Progressive Reduction of TBI Dose fully MHC mismatched T (BALB/c --> B6) minor Hag mismatched T (BALB.B --> B6) From 11 Gy to 0 Gy TBI TCD (10x10^6) ± Donor Th2 Cells Long-term Engraftment Day -2 Day 0 Day 90 % Donor 40 30 20 10 SAHA or JAK3 Inhibitor Preliminary Data Day 7/15/21 Fate of Allospecific T cells - Vβ8+ T cells - CD8(+)H60(TCR+) T cells CP-690,550 is >10000 more potent than AG490 at inhibiting JAK3 0 XRT 900 cgy XRT 900cGy + AG490
Conclusions (Clinical Relevance) Aim 1: understand the biology of graft rejection and develop new target strategies (such as STAT molecule blockade) Aim 2: investigate the possible interaction of STAT6 and STAT1 in determining Th1 and Th2 polarization. Aim3: design new conditioning regimens for non-myeloablative transplants by including drugs that can both: facilitate engraftment (immunomodulatory activity), reduce side effects (specific immune-system activity), control tumor growth (SAHA inhibition of hystone deacetylases that are implicated in several tumors; CP-690,550 is also effective on JAK2 signaling that is implicated in MDS, PV, MF)