Phase I study of azacitidine following donor lymphocyte infusion for relapsed acute myeloid leukemia post allogeneic stem cell transplantation.
ABSTRACT: Donor lymphocyte infusion (DLI) without prophylactic immunosuppression has been used for relapsed AML after allogeneic stem cell transplant (allo-SCT). However DLI is associated with an increased incidence of acute Graft vs. Host Disease (aGVHD). In mice, administration of azacitidine (AzaC) on days 4, 6, 8, and 10 post DLI increases regulatory T cell (Treg) numbers and prevents GVHD without hindering Graft vs. Leukemia (GVL). Based on these findings, we conducted a phase 1 study of AzaC post DLI for AML relapse post allo-SCT. AzaC was administered on days 4, 6, 8 and 10 post-DLI. Dose escalation was done using a 3+3 design with three AzaC dose levels: 30mg/m(2) (level -1), 45mg/m(2) (level 1) and 75mg/m(2) (level 2). Three patients were treated in the 45mg/m(2) dose level and 5 patients were treated in the 75mg/m(2) dose level; no DLTs or grade 3-5 treatment related toxicities were observed. After a median follow-up of 5.2 months, no patients developed grade III-IV aGVHD and no patients died of aGVHD. Six out of 8 patients in the treatment group responded to treatment including two cytogenetic complete remissions, one hematologic complete remission, and three complete remissions with incomplete count recovery. In conclusion, administration of AzaC early post DLI is well tolerated and can potentially prevent GVHD after DLI. Further studies are required to evaluate the effect of azacitidine early post DLI on GVHD and GVL.
Project description:Background:Successful prevention of post-transplantation relapse after donor lymphocyte infusion (DLI) depends on its capability to mediate an effective graft-versus-leukemia (GVL) response while minimizing DLI-related toxicity, including graft-versus-host disease (GVHD). Methods:We assessed the effects of decitabine (DEC), a hypomethylating agent, upon allogeneic immune reaction in a murine model of DLI. Results:Significantly greater tumor growth retardation and survival prolongation occurred in mice administered with 1.0 mg/kg DEC for 5 days (DEC-1.0) than in control or DEC-0.1 mice. Upon prompt DEC and DLI co-administration, dendritic cells (DCs) were activated; DEC-1.0/DLI induced severe GVHD, and survival was significantly lower than with DLI alone or DEC-0.1/DLI treatments. IFN-? and CD28 levels were higher in splenic DCs of DEC-1.0 mice than in those of control mice. Assessment of delayed DLI co-administration with DEC, when IFN-? levels were normalized to control levels, revealed that DEC-1.0/DLI successfully facilitated tumor management without causing severe GVHD. Conclusions:Our results suggest that DEC primes allogeneic immune reactions of DLI via DC activation, and GVHD and GVL effects are separable through optimal DLI timing based on DEC-induced increase in IFN-? expression levels.
Project description:Regulatory T cells (Tregs) suppress graft-versus-host disease (GVHD) while preserving a beneficial graft-versus-leukemia (GVL) effect. Thus, their use in allogeneic stem cell transplantation (SCT) provides a promising strategy to treat GVHD. However, 3 obstacles prevent their routine use in human clinical trials: (1) low circulating number of Tregs in peripheral blood, (2) loss of suppressor function after in vitro expansion, and (3) lack of Treg-specific surface markers necessary for efficient purification. FOXP3 is exclusively expressed in Tregs and forced expression in CD4(+)CD25(-) T cells can convert these non-Tregs into Tregs with functional suppressor function. Here, we show that the FDA-approved hypomethylating agents, decitabine (Dec) and azacitidine (AzaC), induce FOXP3 expression in CD4(+)CD25(-) T cells both in vitro and in vivo. Their suppressor function is dependent on direct contact, partially dependent on perforin 1 (Prf1), but independent of granzyme B (GzmB), and surprisingly, Foxp3. Independence of Foxp3 suggests that genes responsible for the suppressor function are also regulated by DNA methylation. We have identified 48 candidate genes for future studies. Finally, AzaC treatment of mice that received a transplant of major histocompatibility complex mismatched allogeneic bone marrow and T cells mitigates GVHD while preserving GVL by peripheral conversion of alloreactive effector T cells into FOXP3(+) Tregs and epigenetic modulation of genes downstream of Foxp3 required for the suppressor function of Tregs.
Project description:Patients with leukemia who receive a T cell-depleted allogeneic stem cell graft followed by postponed donor lymphocyte infusion (DLI) can experience graft-versus-leukemia (GVL) reactivity, with a lower risk of graft-versus-host disease (GVHD). Here, we have investigated the magnitude, diversity, and specificity of alloreactive CD8 T cells in patients who developed GVL reactivity after DLI in the absence or presence of GVHD. We observed a lower magnitude and diversity of CD8 T cells for minor histocompatibility antigens (MiHAs) in patients with selective GVL reactivity without GVHD. Furthermore, we demonstrated that MiHA-specific T cell clones from patients with selective GVL reactivity showed lower reactivity against nonhematopoietic cells, even when pretreated with inflammatory cytokines. Expression analysis of MiHA-encoding genes showed that similar types of antigens were recognized in both patient groups, but in patients who developed GVHD, T cell reactivity was skewed to target broadly expressed MiHAs. As an inflammatory environment can render nonhematopoietic cells susceptible to T cell recognition, prevention of such circumstances favors induction of selective GVL reactivity without development of GVHD.
Project description:Among hematologic neoplasms, chronic myeloid leukemia (CML) is exquisitely sensitive to graft-versus-leukemia (GVL) because patients relapsing after allogeneic hematopoietic stem-cell transplantation (alloHSCT) can be cured by donor leukocyte infusion (DLI); however, the cellular mechanisms and strategies to separate GVL from GVHD are unclear. We used a BCR-ABL1 transduction/transplantation mouse model to study the mechanisms of DLI in MHC-matched, minor histocompatibility antigen-mismatched allogeneic chimeras with CML-like leukemia, in which DLI can be administered at the time of transplantation (early) or after recovery of hematopoiesis (delayed). After early DLI, CML-like leukemia cannot be transferred into immunocompetent secondary recipients as soon as 4 days after primary transplantation, demonstrating that cotransplantation of T lymphocytes blocks the engraftment of BCR-ABL1-transduced stem cells. In contrast, in allogeneic chimeras with established CML-like leukemia, combined treatment with delayed DLI and the kinase inhibitor imatinib eradicates leukemia with minimal GVHD. The GVL effect is directed against minor histocompatibility antigens shared by normal and leukemic stem cells, and is mediated predominantly by CD8+ T cells, with minor contributions from CD5- splenocytes, including natural killer cells. These results define a physiologic model of adoptive immunotherapy of CML that will be useful for investigating the cellular and molecular mechanisms of GVL.
Project description:Azacitidine (AzaC) mitigates graft-versus-host disease (GvHD) in both murine preclinical transplant models and in human clinical trials while maintaining a robust graft-versus-leukemia effect. Previous studies have failed to investigate the role of natural regulatory T cells (nTregs) on the mitigation of GvHD by AzaC, instead focusing on the generation of suppressive Tregs (CD4+CD25+FOXP3+) through the in vivo conversion of alloreactive donor T effectors (Teffs; CD4+CD25-FOXP3-) and the direct antiproliferative effects of AzaC on allogeneic T cells. Using B6.Foxp3DTR/GFP mice in which Tregs can be specifically ablated through administration of diphtheria toxin, we demonstrate that natural Tregs are required in the donor graft for AzaC to optimally protect against GvHD and that nTregs, unlike Teffs (CD3+FOXP3-), are resistant to the antiproliferative effects of AzaC. Gene expression analysis identified the potent cell cycle inhibitor, p21, was significantly upregulated in Teffs but not nTregs after treatment with AzaC. Furthermore, we demonstrate that Teffs deficient in p21 are less sensitive to the antiproliferative effects of AzaC. These results demonstrate that nTregs are essential for AzaC to fully protect against GvHD and have important clinical implications for future clinical trials testing AzaC as a novel method of GvHD prophylaxis in man.
Project description:SUMMARY: Allogeneic hematopoietic progenitor cell transplantation (HPCT) is a crucial therapeutic option in hematological malignancies, and the graft-versus-leukemia (GvL) effect builds the cornerstone of a long-lasting remission. Cyto-toxic T cells are known to be the primary effector cells in GvL. They recognize minor histocompatibility antigens (mHags) and tumor/leukemia-associated antigens. In case of disease relapse after HPCT, donor lymphocyte infusion (DLI) is an important treatment option for re-induction of remission. However, both treatments, HPCT and DLI carry the risk of morbidity and mortality due to graft-versus-host disease (GvHD) and severe infections. Therefore, the development of targeted adoptive immunotherapy with a lower risk of GvHD is needed, and several study groups are working on that topic.
Project description:Following bone marrow transplantation, delayed donor leukocyte infusions (DLIs) can induce graft-versus-leukemia (GVL) effects without graft-versus-host disease (GVHD). These antitumor responses are maximized by the presence of host hematopoietic antigen-presenting cells (APCs) at the time of DLI. Using a tumor-protection model, we demonstrate here that GVL activity following administration of DLIs to established mixed chimeras is dependent primarily on reactivity to allogeneic MHC antigens rather than minor histocompatibility or tumor-associated antigens. CD8(+) T-cell-dependent GVL responses against an MHC class II-negative tumor following delayed DLI require CD4(+) T-cell help and are reduced significantly when host APCs lack MHC class II expression. CD4(+) T cells primed by host APCs were required for maximal expansion of graft-versus-host reactive CD8(+) T cells but not their synthesis of IFN-gamma. In contrast, the GVL requirement for CD4(+) T-cell help was bypassed almost completely when DLI was administered to freshly irradiated recipients, indicating that the host environment is a major factor influencing the cellular mechanisms of GVL.
Project description:Allogeneic stem cell transplantation (allo-SCT) is the preferred curative treatment for several hematological malignancies. The efficacy of allo-SCT depends on the graft-versus-leukemia (GvL) effect. However, the prognosis of patients with relapsed acute myeloid leukemia (AML) following allo-SCT is poor. Donor lymphocyte infusion (DLI) is utilized after allo-SCT in this setting to prevent relapse, to prolong progression free survival, to establish full donor chimerism and to restore the GvL effect in patients with hematological malignancies. Thus, there are different options for the administration of DLI in AML patients. DLI is currently used prophylactically and in the setting of an overt relapse. In addition, in the minimal residual disease (MRD) setting, DLI may be a possibility to improve overall survival. However, DLI might increase the risk of severe life-threatening complications such as graft-versus-host disease (GvHD) as well as severe infections. The transfusion of lymphocytes has been tested not only for the treatment of hematological malignancies but also chronic infections. In this context, manipulated DLI in a prophylactic or therapeutic approach are an option, e.g., virus-specific DLI using different selection methods or antigen-specific DLI such as peptide-specific CD8+ cytotoxic T lymphocytes (CTLs). In addition, T cells are also genetically engineered, using both chimeric antigen receptor (CAR) genetically modified T cells and T cell receptor (TCR) genetically modified T cells. T cell therapies in general have the potential to enhance antitumor immunity, augment vaccine efficacy, and limit graft-versus-host disease after allo-SCT. The focus of this review is to discuss the different strategies to use donor lymphocytes after allo-SCT. Our objective is to give an insight into the functional effects of DLI on immunogenic antigen recognition for a better understanding of the mechanisms of DLI. To ultimately increase the GvL potency without raising the risk of GvHD at the same time.
Project description:Azacitidine (AzaC) mitigates Graft vs. Host Disease (GVHD) in both murine preclinical transplant models and in human clinical trials while maintaining a robust Graft vs. Leukemia (GVL) effect. Previous studies have failed to investigate the role of natural Tregs on the mitigation of GVHD by AzaC; instead focusing on the generation of suppressive regulatory T cells (Tregs, CD4+CD25+FOXP3+) through the in vivo conversion of alloreactive donor T effectors (Teff CD4+ CD25-, FOXP3-) and the direct anti-proliferative effects of AzaC on allogeneic T cells. Using B6.Foxp3DTR/GFP mice in which Tregs can be specifically ablated through administration of Diphtheria toxin, we demonstrate that nTregs are required in the donor graft for AzaC to optimally protect against GvHD and that nTregs, unlike T effectors (Teff, CD3+FOXP3-), are resistant to the anti-proliferative effects of AzaC. Gene expression analysis identified the potent cell cycle inhibitor, p21, was significantly upregulated in Teff but not nTregs after treatment with AzaC. Furthermore, we demonstrate that Teff cells deficient in p21 are less sensitive to the antiproliferative effects of AzaC. These results, demonstrate that nTregs are essential for AzaC to fully protect against GvHD and have important clinical implications for future clinical trials testing AzaC as a novel method of GVHD prophylaxis in man. Overall design: n=3 per group nTregs and AzaC generated Tregs, nTregs were purified from the spleens of B6.Foxp3GFP mice and Treg depleted T cells were obtained from B6. Foxp3GFP x B6.CAGDSRED. T Cells were co-cultured at a 1:10 ratio of nTreg to Teff and were activated for 2 days in the presence of anti-CD3/CD28 beads (bead:cell 1:1; Invitrogen) and Xcyte medium supplemented with L-glutamine (4mM), penicillin (100 U/mL), streptomycin (100 µg/mL), and human recombinant IL-2 (hIL-2; 500 U/mL). The activated T cells were incubated in the presence of AzaC (1µM) (Sigma-Aldrich) or PBS for an additional 2 days. Cells were sorted using FACS Aria II (BD) to isolate nTreg (CD4+DSRED-FOXP3GFP+), CD4+ Teff (CD4+DSRed+FOXP3GFP-), CD8+ Teff (CD8+DSRed+FOXP3GFP-).
Project description:Graft-versus-host disease (GvHD) remains a significant complication of allogeneic hematopoietic cell transplantation (HCT), associated with significant morbidity and mortality. GvHD is characterized by dysregulated immune responses and resulting tissue damage of target organs. Recent investigations have focused on Foxp3+ regulatory T cells (Tregs) as a therapeutic tool, based on its regulatory functions in GvHD pathogenesis and their instrumental role in mitigating GvHD severity while preserving graft-versus-leukemia (GvL) activity. There are several challenges to its clinical application, including their paucity, impaired suppressive activity, and instability in vivo. Herein, we report that IL-27 pre-stimulation enhances suppressive functions of both mouse and human Tregs. In a complete MHC mismatched murine bone marrow transplant model, IL-27 pre-stimulated polyclonal iTregs diminish acute (a)GvHD lethality, while preserving the GvL effect. Allo-antigen specificity further improves suppressive functions when combined with IL-27 pre-stimulation. In a xenogeneic (human to mouse) GvHD model, IL-27 pre-stimulated human iTregs are superior in protecting recipients from GvHD. Lastly, we compared gene expression profiles of circulating Tregs isolated from HCT recipients with and without aGvHD and found that Tregs from aGvHD patients express distinct gene signatures enriched in immune activation and inflammation. Therefore, these results highlight a novel function of IL-27 in enforcing Treg functions to prevent aGvHD mediated lethality, proposing the hypothesis that dysregulated Treg functions may account for the potential mechanisms underlying GvHD development.