Expansion of CD56-negative, CD16-positive, KIR-expressing natural killer cells after T cell-depleted haploidentical hematopoietic stem cell transplantation.

The main functions of natural killer (NK) cells are early protection against viruses or tumor cells and production of cytokines that regulate immune functions. The present study assessed the role of different NK subsets in exerting graft-versus-leukemia effects in recipients of human leukocyte antigen (HLA) haploidentical hematopoietic transplants and monitored for the first time CD3-/CD56- lymphocyte expansion. CD3-/CD56- cells expressed NK cell-associated molecules, such as CD16, NKp46, NKp30, CD244 (2B4), CD161, and killer cell immunoglobulin-like receptors. CD3-/CD56- cells further exhibited the classical functional characteristics of NK cells: cytolysis of target cells lacking HLA class I, antibody-dependent cellular cytotoxicity and cytokine production. These results demonstrate that CD56- NK cells are functional, recognize missing self and, like their CD56+ counterparts, may contribute to graft-versus-leukemia reactions.

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.

Human leukocyte antigens A23, A24, and A32 but not A25 are ligands for KIR3DL1.

Inhibitory killer cell immunoglobulin receptors (KIR) bind to major histocompatibility complex antigens. Concise knowledge of KIR ligands allows prediction of natural killer (NK)-cell alloreactivity after hematopoietic stem cell transplantation. KIR3DL1 binds to the Bw4 epitope on HLA-B antigens. Although the same epitope is also found on 4 HLA-A antigens (HLA-A23/24/25/32), these are not currently regarded as KIR3DL1 ligands. We show that expression of HLA A*2301, A*2402, or A*3201 but not HLA A*2501 protects target cells from lysis by KIR3DL1(+) NK cells. KIR3DL1(+) NK cells from donors expressing the Bw4 epitope on an HLA-A antigen only are fully functional and capable of lysing Bw4(-) target cells. HLA A25 differs at amino acid 90, close to the serologic Bw4 epitope, from A23/24/32 and from Bw4(+) HLA-B antigens. These data suggest that HLA-A antigens should be taken into consideration when assessing the potential for NK alloreactivity after hematopoietic stem cell transplantation.

NK cell alloreactivity and allogeneic hematopoietic stem cell transplantation.

As only 60% of leukaemia patients find a matched donor, the Perugia Bone Marrow Transplant Centre developed transplantation from HLA haplotype-mismatched family donors to provide a cure for more patients [F. Aversa, A. Tabilio, A. Terenzi, et al., Successful engraftment of T-cell-depleted haploidentical "three-loci" incompatible transplants in leukemia patients by addition of recombinant human granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells to bone marrow inoculum, Blood 84 (1994) 3948-3955] [F. Aversa, A. Tabilio, A. Velardi, et al., Treatment of high-risk acute leukemia with T-cell-depleted stem cells from related donors with one fully mismatched HLA haplotype, N. Engl. J. Med. 339 (1998) 1186-1193] [F. Aversa, A. Terenzi, A. Tabilio, et al., Full haplotype-mismatched hematopoietic stem-cell transplantation: a phase II study in patients with acute leukemia at high risk of relapse, J. Clin. Oncol. 23 (2005) 3447-3454]. HLA-mismatches trigger donor vs. recipient NK cell alloreactivity which improves engraftment, protects from GvHD and reduces relapse in AML patients [L. Ruggeri, M. Capanni, E. Urbani, et al., Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants, Science 295 (2002) 2097-2100], [L. Ruggeri, A. Mancusi, M. Capanni, E. Urbani, A. Carotti, T. Aloisi, M. Stern, D. Pende, K. Perruccio, E. Burchielli, F. Topini, E. Bianchi, F. Aversa, M.F. Martelli, A. Velardi, Donor natural killer cell allorecognition of missing self in haploidentical hematopoietic transplantation for acute myeloid leukemia: challenging its predictive value, Blood, in press]. We are using murine transplant models to determine whether NK cell alloreactivity can be exploited to reduce transplant-related mortality (TRM) which remains a major issue. Data from these on-going studies show pre-transplant infusion of alloreactive NK cells: (1) ablates AML cells, (2) kills recipient T cells, permitting a reduced toxicity conditioning regimen, and (3) ablates the recipient dendritic cells (DCs) which trigger GvHD, thus protecting from GvHD while permitting a higher T cell content in the graft. We are designing a clinical haploidentical transplant trial using alloreactive NK cells in the conditioning regimen, with the aim of reducing TRM and improving outcomes and overall survival.

Microparticles derived from endothelial progenitor cells in patients at different cardiovascular risk.

OBJECTIVES: Exposure to cardiovascular risk factors causes the release of pro-atherogenic microparticles from vascular cells and reduces the number of the atheroprotective endothelial progenitor cells (EPCs). We investigated whether microparticles shedding from EPCs are detectable in cultures of EPCs and in the circulation of subjects with various degrees of cardiovascular risk. We also investigated the relationship of EPCs-derived microparticles to cardiovascular risk factors and aortic stiffness, a marker of cardiovascular risk and impaired vascular repair by EPCs. METHODS AND RESULTS: We estimated the 10-year Framingham risk score in 105 individuals with various degrees of cardiovascular risk and measured the number of circulating EPCs, EPCs-derived microparticles (CD34+/KDR+) and aortic stiffness. Release of CD34+/KDR+ microparticles was tested in cultures of EPCs exposed to hydrogen-peroxide. CD34+/KDR+ microparticles were found in EPCs cultures incubated with hydrogen-peroxide. Framingham risk was associated with EPCs (r=-0.47, p<0.001) and CD34+/KDR+ microparticles (r=0.56, p<0.001). Low EPCs (r=-0.59, p<0.001) and high CD34+/KDR+ microparticle (r=0.57, p<0.001) levels were predictors of aortic stiffness, independent of the Framingham risk. CONCLUSIONS: EPCs undergo fragmentation into microparticles when exposed to a pro-apoptotic milieu. Increased microparticle shedding from EPCs may reduce circulating EPCs levels and may thus contribute to increase aortic stiffness beside traditional risk factors.

Donor natural killer cell allorecognition of missing self in haploidentical hematopoietic transplantation for acute myeloid leukemia: challenging its predictive value.

We analyzed 112 patients with high-risk acute myeloid leukemia (61 in complete remission [CR]; 51 in relapse), who received human leukocyte-antigen (HLA)-haploidentical transplants from natural killer (NK) alloreactive (n = 51) or non-NK alloreactive donors (n = 61). NK alloreactive donors possessed HLA class I, killer-cell immunoglobulin-like receptor (KIR) ligand(s) which were missing in the recipients, KIR gene(s) for missing self recognition on recipient targets, and alloreactive NK clones against recipient targets. Transplantation from NK-alloreactive donors was associated with a significantly lower relapse rate in patients transplanted in CR (3% versus 47%) (P > .003), better event-free survival in patients transplanted in relapse (34% versus 6%, P = .04) and in remission (67% versus 18%, P = .02), and reduced risk of relapse or death (relative risk versus non-NK-alloreactive donor, 0.48; 95% CI, 0.29-0.78; P > .001). In all patients we tested the "missing ligand" model which pools KIR ligand mismatched transplants and KIR ligand-matched transplants from donors possessing KIR(s) for which neither donor nor recipient have HLA ligand(s). Only transplantation from NK-alloreactive donors is associated with a survival advantage.

Increased ratio of CD31+/CD42- microparticles to endothelial progenitors as a novel marker of atherosclerosis in hypercholesterolemia.

OBJECTIVE: Atherosclerosis may be caused by increased endothelial damage and by a consumptive loss of endothelial repair capacity by endothelial progenitors. Arterial stiffness is a reliable marker of atherosclerosis and a positive correlate of endothelial damage. We investigated whether an increased ratio of CD31+/CD42- microparticles to endothelial progenitors, a possible indicator of endothelial damage and impaired endothelium reparation, may contribute to aortic stiffness in hypercholesterolemia. We also studied the in vitro effect of microparticles from hypercholesterolemic patients on endothelial progenitor survival. METHODS AND RESULTS: Circulating CD31+/CD42- microparticles, endothelial progenitors, and aortic pulse wave velocity (aPWV), a measure of aortic stiffness, were measured in 50 patients with never-treated hypercholesterolemia and 50 normocholesterolemic controls. Hypercholesterolemic patients had more circulating CD31+/CD42- microparticles, less endothelial progenitors, and a stiffer aorta than controls. aPWV was associated with CD31+/CD42- microparticles (r=0.61; P<0.001), endothelial progenitors (r=-0.45, P<0.001), and with cholesterol levels (r=0.51; P<0.001). High plasma cholesterol and a high ratio of CD31+/CD42- microparticles to endothelial progenitors independently predicted an increased aPWV. Microparticles from hypercholesterolemic patients caused a significant endothelial progenitor loss in vitro. CONCLUSIONS: Hypercholesterolemia-related aortic stiffness is promoted by plasma cholesterol directly, increased endothelial damage, and reduced endothelium repair capacity by endothelial progenitors.

Transferring functional immune responses to pathogens after haploidentical hematopoietic transplantation.

Aspergillus and cytomegalovirus are major causes of morbidity/mortality after haploidentical hematopoietic transplantation. The high degree of mismatching makes cell immunotherapy impossible as it would result in lethal graft-versus-host disease (GvHD). We generated large numbers of donor T-cell clones specific for Aspergillus or cytomegalovirus antigens. We identified clones potentially responsible for causing GvHD by screening them for cross-reactivity against recipient mononuclear cells. Non-recipient reactive, pathogen-specific clones were infused soon after transplantation. They were CD4+ and produced high levels of interferon-gamma and low levels of interleukin-10. In 46 control transplant recipients who did not receive adoptive therapy, spontaneous pathogen-specific T cells occurred in low frequency 9 to 12 months after transplantation and displayed a non-protective low interferon-gamma/high interleukin-10 production phenotype. In the 35 recipients who received adoptive therapy, one single infusion of donor alloantigen-deleted, pathogen-specific clones in the dose range of 10(5) to 10(6) cells/kg body weight did not cause GvHD and induced high-frequency T-cell responses to pathogens, which exhibited a protective high interferon-gamma/low interleukin-10 production phenotype within 3 weeks of infusion. Frequencies of pathogen-specific T cells remained stable over time, and were associated with control of Aspergillus and cytomegalovirus antigenemia and infectious mortality. This study opens new perspectives for reducing infectious mortality after haploidentical transplantations.

The impact of donor natural killer cell alloreactivity on allogeneic hematopoietic transplantation.

Although natural killer (NK) cells are triggered to kill by many activating receptors, lysis of autologous cells is blocked by inhibitory receptors (called Killer cell Ig-like receptors or KIRs) which recognize epitopes shared by certain major histocompatibility complex (MHC) class I allele groups (called KIR ligands). As these inhibitory receptors are clonally distributed, they constituted a repertoire containing different allospecificities. Thus, the NK cells in the repertoire are lytic against allogeneic targets that do not express their inhibitory KIR ligands. In hematopoietic human-leukocyte-antigen (HLA)-haplotype mismatched transplantation, donor-vs-recipient alloreactive NK cells improve engraftment, decrease the incidence of leukemia relapse and do not cause Graft-vs-Host disease (GvHD). Pre-transplant molecular high-resolution HLA of recipient and donor, KIR genotyping of the donor and direct assessment of the donor NK repertoire identify donors with the potential for donor-vs-recipient NK cell alloreactivity.

Exploitation of alloreactive NK cells in adoptive immunotherapy of cancer.

NK cells are primed to kill by several activating receptors. Killing of autologous cells is prevented as NK cells co-express inhibitory receptors for self-MHC class I molecules. Human NK cells discriminate between different allelic forms of MHC molecules via killer cell immunoglobulin-like receptors (KIRs), which are clonally distributed, and each cell in the repertoire bears at least one receptor that is specific for self-MHC class I molecules. Consequently, when faced with mismatched allogeneic targets, NK cells in the repertoire will sense the missing expression of self-MHC class I alleles and will mediate alloreactions. Recent studies in murine transplant models and data from mismatched haematopoietic transplant trials demonstrate MHC class I mismatches, which generate an alloreactive NK-cell response in the graft-versus-host direction, eradicate leukaemia, improve engraftment and protect against T-cell-mediated graft-versus-host disease.

Natural killer cell alloreactivity in haploidentical hematopoietic stem cell transplantation.

Natural killer (NK) cells are primed to kill by several activating receptors. NK cell killing of autologous cells is prevented because NK cells coexpress inhibitory receptors (killer cell immunoglobulin-like receptors [KIR]) that recognize groups of (self) major histocompatibility complex class I alleles. Because KIRs are clonally distributed, the NK cell population in any individual are constituted of a repertoire with a variety of class I specificities. NK cells in the repertoire mediate alloreactions when the allogeneic targets do not express the class I alleles that block them. After haploidentical hematopoietic transplantation, NK cell-mediated donor-versus-recipient alloresponses reduce the risk of relapse in acute myeloid leukemia patients while improving engraftment and protecting against graft-versus-host disease. High-resolution molecular HLA typing of recipient and donor, positive identification of donor KIR genes, and, in some cases, functional assessment of donor NK clones identify haploidentical donors who are able to mount donor-versus-recipient NK alloreactions.

Alloreactive natural killer cells in mismatched hematopoietic stem cell transplantation.

Natural killer (NK) cell-mediated, donor-vs.-recipient alloresponses occur following transplantation of human leukocyte antigen (HLA) haplotype-mismatched hematopoietic stem cells (HSCs). NK cell alloreactivity reduced the risk of relapse in acute myeloid leukemia patients while improving engraftment and protecting against graft-vs.-host disease (GvHD). NK cells are primed to kill by several activating receptors. NK killing of autologous cells is prevented because NK cells co-express inhibitory receptors (killer cell Ig-like receptors, KIR) that recognize groups of (self) MHC class I alleles. As KIRs are clonally distributed, the NK population in any individual is constituted of a repertoire with different allospecificities. NK cells in the repertoire mediate alloreactions when the allogeneic targets do not express class I alleles that block them. High resolution molecular HLA typing of recipient and donor, positive identification of donor KIR genes, and in some cases, functional assessment of donor NK clones will identify haploidentical donors who are able to mount donor-vs.-recipient NK alloreactions.

Natural killer cells as a therapeutic tool in mismatched transplantation.

Natural-killer-cell-mediated, donor-vs-recipient alloresponses occur following transplantation of human-leukocyte-antigen (HLA)-haplotype-mismatched haematopoietic stem cells. Natural killer (NK) cell alloreactivity reduces the risk of relapse in acute myeloid leukaemia patients, while improving engraftment and protecting against graft-vs-host disease. NK cells are primed to kill by several activating receptors. NK cell killing of autologous cells is prevented as NK cells co-express inhibitory receptors (killer cell Ig-like receptors, KIR) that recognize groups of (self) major histocompatibility complex class I alleles. As KIRs are distributed clonally, the NK cell population in any individual constitutes a repertoire with different allospecificities. NK cells in the repertoire mediate alloreactions when the allogeneic targets do not express class I alleles that block them. High-resolution molecular HLA typing of recipient and donor, positive identification of donor KIR genes and, in some cases, functional assessment of donor NK clones will identify haplo-identical donors who are able to mount donor-vs-recipient NK alloreactions.

Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants.

T cells that accompany allogeneic hematopoietic grafts for treating leukemia enhance engraftment and mediate the graft-versus-leukemia effect. Unfortunately, alloreactive T cells also cause graft-versus-host disease (GVHD). T cell depletion prevents GVHD but increases the risk of graft rejection and leukemic relapse. In human transplants, we show that donor-versus-recipient natural killer (NK)-cell alloreactivity could eliminate leukemia relapse and graft rejection and protect patients against GVHD. In mice, the pretransplant infusion of alloreactive NK cells obviated the need for high-intensity conditioning and reduced GVHD. NK cell alloreactivity may thus provide a powerful tool for enhancing the efficacy and safety of allogeneic hematopoietic transplantation.