Modulating STAT Signaling to Promote Engraftment of Allogeneic Bone Marrow Transplant

Transcription

1 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

2 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

3 Role of STAT Signaling in Immunologic Response Laurence, Nat Immunol 2007

4 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

5 Graft rejection Model: TBI and Host T Cell Add-back (Reisner, Blood 2003; Mariotti, JI 2009) 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)

6 Th1/Tc1 >> Th2/Tc2 for mediating Graft Rejection vs. P<0.0005, vs. P< B6 BALB/c Donor alone Host T Th1/Tc1 Th2/Tc2 Survival (%) Time after T (d) Percent Donor alone + Th2/Tc2

7 STAT1 signaling is Indispensable for Graft Rejection BALB/c B6 100 vs. P<.01 Donor only Host Stat1 -/- T WT T Survival (%) Chimerism-d Time after T (d) 100 Percent Donor Spleen Blood Spleen Blood only + Stat1 -/- T

8 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 ) * * * * * * * * * * * * 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

9 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

10 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 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 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

12 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

13 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)

14 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

15 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

16 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.

17 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 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

18 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)