Loredana Ruggeri

Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation

Hastening posttransplantation immune reconstitution is a key challenge in human leukocyte antigen (HLA)-haploidentical hematopoietic stem-cell transplantation (HSCT). In experimental models of mismatched HSCT, T-regulatory cells (Tregs) when co-infused with conventional T cells (Tcons) favored posttransplantation immune reconstitution and prevented lethal graft-versus-host disease (GVHD). In the present study, we evaluated the impact of early infusion of Tregs, followed by Tcons, on GVHD prevention and immunologic reconstitution in 28 patients with high-risk hematologic malignancies who underwent HLA-haploidentical HSCT. We show for the first time in humans that adoptive transfer of Tregs prevented GVHD in the absence of any posttransplantation immunosuppression, promoted lymphoid reconstitution, improved immunity to opportunistic pathogens, and did not weaken the graft-versus-leukemia effect. This study provides evidence that Tregs are a conserved mechanism in humans.

Full Haplotype-Mismatched Hematopoietic Stem-Cell Transplantation: A Phase II Study in Patients With Acute Leukemia at High Risk of Relapse

PURPOSE Establishment of hematopoietic stem-cell (HSC) transplantation from mismatched relatives is feasible for patients with acute leukemia. As our original method of graft processing was unsuitable for large-scale clinical studies, we use automated devices for CD34+ cell purification. PATIENTS AND METHODS Sixty-seven patients with acute myeloid leukemia (AML; 19 complete remission [CR] 1, 14 CR 2, nine CR > 2, 25 in relapse) and 37 with acute lymphoid leukemia (ALL; 14 CR 1, eight CR 2, two CR > 2, 13 in relapse) were conditioned with total-body irradiation, thiotepa, fludarabine, and antithymocyte globulin. Peripheral-blood progenitor cells were mobilized with recombinant human granulocyte colony-stimulating factor and depleted of T-cells using CD34+ cell immunoselection. No post-transplantation graft-versus-host disease (GvHD) prophylaxis was administered. RESULTS Primary engraftment was achieved in 94 of 101 assessable patients. Six of the seven patients who rejected the primary graft, engrafted after a second transplantation. Overall, 100 of 101 patients engrafted. Acute GvHD developed in eight of 100 patients, and chronic GvHD, in five of 70 assessable patients. Thirty-eight patients died of nonleukemic causes. Relapse occurred in nine of 66 patients receiving transplantation in remission and in 17 of 38 receiving transplantation in relapse. Median follow-up of the 40 patients who survived event-free was 22 months (range, 1 to 65 months). Event-free survival (+/- standard deviation) rate was 48% +/- 8% and 46% +/- 10%, respectively, for the 42 AML and 24 ALL patients receiving transplantation in remission. CONCLUSION Our transplantation procedure provides reliable, reproducible CD34+ cell purification, high engraftment rates, and prevention of GvHD. The mismatched-related transplant emerges as a viable, alternative source of stem cells for acute leukemia patients without matched donors and/or those who urgently need transplantation.

Preclinical characterization of 1-7F9, a novel human anti―KIR receptor therapeutic antibody that augments natural killer―mediated killing of tumor cells

Inhibitory-cell killer immunoglobulin-like receptors (KIR) negatively regulate natural killer (NK) cell-mediated killing of HLA class I-expressing tumors. Lack of KIR-HLA class I interactions has been associated with potent NK-mediated antitumor efficacy and increased survival in acute myeloid leukemia (AML) patients upon haploidentical stem cell transplantation from KIR-mismatched donors. To exploit this pathway pharmacologically, we generated a fully human monoclonal antibody, 1-7F9, which cross-reacts with KIR2DL1, -2, and -3 receptors, and prevents their inhibitory signaling. The 1-7F9 monoclonal antibody augmented NK cell-mediated lysis of HLA-C-expressing tumor cells, including autologous AML blasts, but did not induce killing of normal peripheral blood mononuclear cells, suggesting a therapeutic window for preferential enhancement of NK-cell cytotoxicity against malignant target cells. Administration of 1-7F9 to KIR2DL3-transgenic mice resulted in dose-dependent rejection of HLA-Cw3-positive target cells. In an immunodeficient mouse model in which inoculation of human NK cells alone was unable to protect against lethal, autologous AML, preadministration of 1-7F9 resulted in long-term survival. These data show that 1-7F9 confers specific, stable blockade of KIR, boosting NK-mediated killing of HLA-matched AML blasts in vitro and in vivo, providing a preclinical basis for initiating phase 1 clinical trials with this candidate therapeutic antibody.

NCRs and DNAM-1 mediate NK cell recognition and lysis of human and mouse melanoma cell lines in vitro and in vivo

NK cells use a variety of receptors to detect abnormal cells, including tumors and their metastases. However, in the case of melanoma, it remains to be determined what specific molecular interactions are involved and whether NK cells control metastatic progression and/or the route of dissemination. Here we show that human melanoma cell lines derived from LN metastases express ligands for natural cytotoxicity receptors (NCRs) and DNAX accessory molecule-1 (DNAM-1), two emerging NK cell receptors key for cancer cell recognition, but not NK group 2 member D (NKG2D). Compared with cell lines derived from metastases taken from other anatomical sites, LN metastases were more susceptible to NK cell lysis and preferentially targeted by adoptively transferred NK cells in a xenogeneic model of cell therapy. In mice, DNAM-1 and NCR ligands were also found on spontaneous melanomas and melanoma cell lines. Interference with DNAM-1 and NCRs by antibody blockade or genetic disruption reduced killing of melanoma cells. Taken together, these results show that DNAM-1 and NCRs are critical for NK cell-mediated innate immunity to melanoma cells and provide a background to design NK cell-based immunotherapeutic strategies against melanoma and possibly other tumors.

Successful transfer of alloreactive haploidentical KIR ligand-mismatched natural killer cells after infusion in elderly high risk acute myeloid leukemia patients

Thirteen patients with acute myeloid leukemia, 5 with active disease, 2 in molecular relapse, and 6 in morphologic complete remission (CR; median age, 62 years; range, 53-73 years) received highly purified CD56(+)CD3(-) natural killer (NK) cells from haploidentical killer immunoglobulin-like receptor-ligand mismatched donors after fludarabine/cyclophosphamide immunosuppressive chemotherapy, followed by IL-2. The median number of infused NK cells was 2.74 × 10(6)/Kg. T cells were < 10(5)/Kg. No NK cell-related toxicity, including GVHD, was observed. One of the 5 patients with active disease achieved transient CR, whereas 4 of 5 patients had no clinical benefit. Both patients in molecular relapse achieved CR that lasted for 9 and 4 months, respectively. Three of 6 patients in CR are disease free after 34, 32, and 18 months. After infusion, donor NK cells were found in the peripheral blood of all evaluable patients (peak value on day 10). They were also detected in BM in some cases. Donor-versus-recipient alloreactive NK cells were shown in vivo by the detection of donor-derived NK clones that killed recipients targets. Adoptively transferred NK cells were alloreactive against recipients cells, including leukemia. In conclusion, infusion of purified NK cells is feasible in elderly patients with high-risk acute myeloid leukemia. This trial was registered at www.clinicaltrial.gov as NCT00799799.

Survival after T cell–depleted haploidentical stem cell transplantation is improved using the mother as donor

We hypothesized that transplacental leukocyte trafficking during pregnancy, which induces long-term, stable, reciprocal microchimerism in mother and child, might influence outcome of patients with acute leukemia given parental donor haploidentical hematopoietic stem cell transplantation (HSCT). We analyzed the outcome of 118 patients who received transplants for acute leukemia in 2 centers. Patients received highly T cell-depleted haploidentical grafts after myelo-ablative conditioning. Five-year event-free survival was better in patients who received transplants from the mother than from the father (50.6% +/- 7.6% vs 11.1% +/- 4.2%; P < .001). Better survival was the result of both reduced incidence of relapse and transplantation-related mortality. The protective effect was seen in both female and male recipients, in both lymphoid and myeloid diseases; it was more evident in patients receiving transplants in remission than in chemotherapy-resistant relapse. Incidences of rejection and acute graft-versus-host disease were not significantly influenced. Multivariate analysis confirmed donor sex in parental donor transplantation as an independent prognostic factor for survival (hazard ratio, father vs mother = 2.36; P = .003). In contrast, in a control cohort of patients who received transplants from haploidentical siblings, donor sex had no influence on outcome. Although obtained in a retrospective analysis, these data suggest that the mother of the patient should be preferred as donor for haploidentical HSCT.

HLA-haploidentical transplantation with regulatory and conventional T-cell adoptive immunotherapy prevents acute leukemia relapse

Posttransplant relapse is still the major cause of treatment failure in high-risk acute leukemia. Attempts to manipulate alloreactive T cells to spare normal cells while killing leukemic cells have been unsuccessful. In HLA-haploidentical transplantation, we reported that donor-derived T regulatory cells (Tregs), coinfused with conventional T cells (Tcons), protected recipients against graft-versus-host disease (GVHD). The present phase 2 study investigated whether Treg-Tcon adoptive immunotherapy prevents posttransplant leukemia relapse. Forty-three adults with high-risk acute leukemia (acute myeloid leukemia 33; acute lymphoblastic leukemia 10) were conditioned with a total body irradiation-based regimen. Grafts included CD34(+) cells (mean 9.7 × 10(6)/kg), Tregs (mean 2.5 × 10(6)/kg), and Tcons (mean 1.1 × 10(6)/kg). No posttransplant immunosuppression was given. Ninety-five percent of patients achieved full-donor type engraftment and 15% developed ≥grade 2 acute GVHD. The probability of disease-free survival was 0.56 at a median follow-up of 46 months. The very low cumulative incidence of relapse (0.05) was significantly better than in historical controls. These results demonstrate the immunosuppressive potential of Tregs can be used to suppress GVHD without loss of the benefits of graft-versus-leukemia (GVL) activity. Humanized murine models provided insights into the mechanisms underlying separation of GVL from GVHD, suggesting the GVL effect is due to largely unopposed Tcon alloantigen recognition in bone marrow.

Allogeneic hematopoietic transplantation and natural killer cell recognition of missing self

Summary:  Although the optimal donor for allogeneic hematopoietic stem cell transplantation (HSCT) is a human leukocyte antigen‐matched sibling, 75% of patients do not have a match, and alternatives are matched unrelated volunteers, unrelated umbilical cord blood units, and full‐haplotype‐mismatched family members. To cure leukemia, allogeneic HSCT relies on donor T cells in the allograft, which promote engraftment, eradicate malignant cells, and reconstitute immunity. Here, we focus on the open issues of rejection, graft‐versus‐host disease (GVHD), and infections and the benefits of natural killer (NK) cell alloreactivity and its underlying mechanisms. Donor‐versus‐recipient NK cell alloreactivity derives from a mismatch between inhibitory receptors for self‐major histocompatibility complex (MHC) class I molecules on donor NK clones and the MHC class I ligands on recipient cells. These NK clones sense the missing expression of the self‐MHC class I allele on the allogeneic targets and mediate alloreactions. HSCT from ‘NK alloreactive’ donors controls acute myeloid relapse without causing GVHD. We review the translation of NK cell recognition of missing self into the clinical practice of allogeneic hematopoietic transplantation and discuss how it has opened innovative perspectives in the cure of leukemia.

Natural killer cell allorecognition of missing self in allogeneic hematopoietic transplantation: a tool for immunotherapy of leukemia

Donor-versus-recipient natural killer (NK) cell alloreactivity has been established as a key therapeutic element in HLA haplotype mismatched hematopoietic transplants in adult AML and pediatric ALL and as a possible beneficial effector in cord blood transplant for AML. It is effected by functional NK cells which express inhibitory killer cell immunoglobulin-like receptor(s) (KIR) for self-class I ligand(s), sense missing expression of donor KIR ligand(s) in the recipient and mediate alloreactions. At present NK cell allotherapy for leukemia is deployed through stem cell transplantation (and ensuing NK cell reconstitution) across KIR ligand mismatches. Studies have been performed to infuse NK cells for immunotherapy outside the fields of transplantation and/or harness the function of endogenous NK cells in patients with hematological malignancies.

Cellular therapy : exploiting NK cell alloreactivity in transplantation

Allogeneic hematopoietic transplantation relies on T-cell alloreactions for engraftment and the graft-versus-leukemia (GVL) effect. In human leukocyte antigen (HLA) haplotype-mismatched transplants, extensive T-cell depletion of the graft is essential to prevent GVHD. This raises the question of whether mismatched transplants exert any GVL effect and whether it will ever be possible to reduce the intensity of preparative regimens. Because natural killer (NK) cells are negatively regulated by MHC class I–specific inhibitory receptors, mismatched transplants may trigger NK-cell alloreactivity. HLA class I disparities driving NK-cell alloreactions in the GVH direction mediate strong GVL effects, produce higher engraftment rates, and do not cause GVHD. In murine MHC-mismatched transplant models with no donor T-cell reactivity against the recipient, the pre-transplant infusion of donor-vs-recipient alloreactive NK cells conditioned the recipients to bone marrow transplantation without GVHD. NK-cell alloreactivity may be a unique therapeutic tool for tolerance induction and clearance of leukemia in hematopoietic transplantation.